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PubMed2002

Assessment of nuclear and cytoplasmic maturation in in-vitro matured human oocytes.

BACKGROUND: With improved prospects for the use of human oocyte in-vitro maturation in assisted reproductive technologies, the need to define more clearly the coordination of nuclear and cytoplasmic maturation has arisen. METHODS: Immunofluorescence and confocal microscopy were used to evaluate cell cycle-dependent modifications in chromatin and microtubules in human germinal vesicle oocytes (n = 455) undergoing in-vitro maturation. RESULTS: Four distinct classes of germinal vesicle stage oocytes were identified based on the expression of G2/interphase characteristics, but, of these, only one class of oocytes was competent to complete meiotic progression to metaphase-II in vitro. The majority of germinal vesicle stage oocytes resumed meiosis within 6 h (88.9%) of culture and exhibited an accelerated pace of progression to metaphase-II (66.7%) over 24 h, but in general were unable to maintain meiotic arrest and defaulted into interphase within 24 h of polar body emission. Characterization of microtubule dynamics and chromatin phosphorylation demonstrates specific cell cycle deficiencies in in-vitro matured human oocytes. CONCLUSION: This work forms a basis for future studies aimed at optimizing nuclear and cytoplasmic maturation during in-vitro maturation.

Biomarkers↗
PubMed2000

Effects of sodium pyruvate in nonserum maturation medium on maturation, fertilization, and subsequent development of bovine oocytes with or without cumulus cells.

The present study was conducted to determine the effects of cumulus cells and sodium pyruvate during in vitro maturation of bovine oocytes on maturation, fertilization, and subsequent development. Cumulus-enclosed oocytes (CEOs) and cumulus-denuded oocytes (CDOs) were cultured for 24 h in polyvinylpyrrolidone-Hepes-tissue culture medium 199 with or without sodium pyruvate. Oocytes were fertilized in vitro and then cultured in CR1aa for 10 days. Before in vitro fertilization, the glutathione (GSH) content of some oocytes was measured. Maturation and normal fertilization rates of CDOs cultured with sodium pyruvate and CEOs were higher than that of CDOs cultured without sodium pyruvate. The CEOs showed significantly higher rates of development to the blastocyst stage than CDOs. The GSH contents of oocytes significantly decreased in CDOs after maturation culture, but the GSH contents of oocytes in CEOs remained at the same level as oocytes before culture. These results indicate that sodium pyruvate promotes nuclear maturation of bovine CDOs and that a continuing presence of cumulus cells during maturation is important for subsequent development of zygotes to the blastocyst stage. However, blastocysts produced from CDOs in the presence of sodium pyruvate showed a developmental competence to be normal calves, but it is not known if CDOs cultured without sodium pyruvate also were capable of developing into calves.

Animals↗
PubMed2005

Growth and maturation of follicles and oocytes following xenotransplantation of porcine ovarian tissues and in vitro maturation.

This is the first report to show morphological evidence of in vitro maturation of oocytes recovered from xenotransplanted antral follicles. To develop a suitable tool for studing the growth and maturation of follicles and oocytes, we xenotransplanted small pieces of ovarian cortical tissue from sows, which contained small preantral follicles (primordial, primary, and secondary follicles; less than 0.05, 0.1 and 0.3 mm in diameter, respectively), under the capsules of kidneys of adult female severe combined immunodeficient (SCID) mice for 2 and 8 weeks, and then recovered cumulus-oocyte complexes from the growing tertiary follicles in xenografted tissues. The distribution of processes from cumulus cells to oocytes and the follicular growth, development, and maturation during xenotransplantation were histochemically analyzed. Tertiary follicles, 0.5 to 3.0 mm in diameter, were obtained from grafted tissues 2 (85%: 52 follicles/61 grafted tissues) and 8 (50%: 15/30) weeks after xenotransplantation, and then oocytes, which were tightly attached to cumulus cells, were collected from each tertiary follicle and cultured to assess their quality. At 2 weeks after grafting, 17.6% of the oocytes had matured to the metaphase II stage, but no such maturation was observed 8 weeks after grafting. Thus, in the 2 weeks group, preantral follicles rapidly grew in xenotransplanted porcine ovarian tissues to the tertiary stage, and oocytes could be recovered and matured from them by in vitro culture.

Actin Cytoskeleton↗
PubMed2005

Comparison of dental maturity in children of different ethnic origins: international maturity curves for clinicians.

Dental maturity was studied with 9577 dental panoramic tomograms of healthy subjects from 8 countries, aged between 2 and 25 years of age. Demirjian's method based on 7 teeth was used for determining dental maturity scores, establishing gender-specific tables of maturity scores and development graphs. The aim of this study was to give dental maturity standards when the ethnic origin is unknown and to compare the efficiency and applicability of this method to forensic sciences and dental clinicians. The second aim was to compare the dental maturity of these different populations. We noted an high efficiency for International Demirjian's method at 99% CI (0.85% of misclassified and a mean accuracy between 2 to 18 years +/- 2.15 years), which makes it useful for forensic purposes. Nevertheless, this international method is less accurate than Demirjian's method developed for a specific country, because of the inter-ethnic variability obtained by the addition of 8 countries in the dental database. There are inter-ethnic differences classified in three major groups. Australians have the fastest dental maturation and Koreans have the slowest.

Adolescent↗
PubMedDate not supplied

[Oocyte maturity and quality: value of intracytoplasmic sperm injection. Fertility of microinjected oocytes after in vitro maturation].

Many studies in IVF practice, have tried to assess the maturity and quality of oocytes prior to insemination and relate it to IVF efficiency and to the pattern of ovarian stimulation. They were based on the indirect evaluation of the aspect of the cumulus-corona-cell complex (CCC) which was rapidly shown to be poorly correlated to the oocyte nuclear and cytoplasmic maturity in stimulated cycles. For sperm microinjection procedures, oocytes need to be peeled off from any follicular cell through hyaluronidase action in order to gain an easy access to the zona pellucida and the ooplasm. If therefore becomes possible to precisely known at recovery the nuclear status of the oocyte cohort as well as the rate of degenerative gametes. Immature oocytes can be further matured in vitro. Moreover, the ICSI procedure (intracytoplasmic sperm injection) allows a direct assessment of the cytoplasmic maturation, whatever the maturity of the ZP and its receptors and of the plasma membrane. On a preliminary evaluation of 70 ICSI cycles performed in our collaborative group from september to november 1994 and leading to a 24 % pregnancy rate per oocyte pick-up we focused on the true maturation of the oocyte cohort, its outcome and correlation with ICSI efficiency and stimulation protocol. On the 760 oocytes, 13.9% were atretic, 9% at the germinal vesicle stage (GV), 3.4% in metaphase 1 and 73.7% in metaphase 2 at recovery.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Antigen processing in populations of mature murine dendritic cells is caused by subsets of incompletely matured cells.

Immature dendritic cells (DC), such as freshly isolated Langerhans cells (LC), are excellent at processing native protein Ag. During short term culture they shut off MHC class II synthesis and down-regulate their processing capacity. They retain, however, the MHC/peptide complexes, up-regulate adhesion and costimulatory molecules, and acquire the ability to sensitize T cells. Two reports describing substantial processing activity in populations of mature DC prompted us to undertake an extensive comparative study of the Ag-processing capacities of immature vs mature DC. We used a panel of 17 peptide-specific T cell hybridomas restricted by six different MHC class II molecules: I-Ab, I-A(d), I-E(d), hybrid I-A beta dE alpha, I-Ak, and I-Ek. Side by side comparisons revealed in all cases that freshly isolated LC were superior to cultured mature LC in their ability to process native proteins. With some hybridomas, however, we found a considerable degree of processing by populations of cultured LC at high doses of Ag or Ag-presenting cells. This activity, however, did not correlate with the MHC haplotype. Direct comparison over wide ranges of DC doses or Ag doses showed that it was always less than that of corresponding fresh immature LC. Immunoperoxidase staining of cytospins and flow cytometry with mAb In1 disclosed a small (20% maximum) subset of cultured LC expressing the MHC class II-associated invariant chain, indicating ongoing biosynthesis of this molecule and, thus, incomplete maturation of these LC. Therefore, the residual processing activity observed in populations of mature DC may be explained by small subpopulations of incompletely matured DC.

Animals↗
PubMed1994

Maturation of human procathepsin B. Proenzyme activation and proteolytic processing of the precursor to the mature proteinase, in vitro, are primarily unimolecular processes.

Recombinant latent human procathepsin B produced in yeast was purified to near homogeneity. The purified recombinant proenzyme is activated in vitro under acidic conditions resulting in rapid conversion into the mature form of the proteinase. Activation as well as proteolytic maturation of the recombinant cathepsin B precursor were shown to be primarily concentration-independent processes indicating a unimolecular (i.e. intramolecular) mechanism. Only one cleavage site was identified, yielding a mature polypeptide with the same amino-terminal sequence as that found in recombinant active human cathepsin B obtained from yeast culture media. The same peptide bond is cleaved during processing of a nonactivatable mutant of procathepsin B by the purified mature enzyme (i.e. intermolecular processing). Thus, the complete proregion is liberated during procathepsin B processing. This peptide may then act as a reversible inhibitor and stabilizer of the mature proteinase, and it appears likely that cathepsin B-propeptide complexes occur transiently during proteolytic maturation.

Amino Acid Sequence↗
PubMed1986

Renal functional maturation: renal handling of proteins by mature and immature newborns.

Renal clearance of creatinine (Ccr), total protein excretion, urinary protein composition and renal clearance of albumin (Calb) were measured and calculated in male premature and mature infants of gestational age 29-41 weeks and in mature infants 1 and 3 months of age. Total protein excretion decreased slightly but not significantly during maturation. The urinary protein composition changed significantly as the fraction of low molecular weight proteins decreased from 48% at a gestational age of 29-33 weeks to 24% in mature infants aged 3 months, the albumin fraction increased from 39%-46% and the proportion of higher molecular weight proteins increased from 12%-29%, respectively. Calb decreased from 2.73-0.80 microliter/min/1.73 m2 in the presence of a rise in Ccr, resulting in a significant fall of the ratio Calb/Ccr from 0.0137 in the youngest prematures to 0.00147 in 3-month-old mature infants.

Albuminuria↗
PubMed1989

Activity of maturation promoting factor in pig oocytes after microinjection and serial transfer of maturing cytoplasm.

Microinjection of 120 pl homologous or heterologous (mouse) maturing cytoplasm into immature pig oocytes resulted in germinal vesicle breakdown (GVBD) and chromosome condensation (CC) after 8 h of culture. In contrast, nearly all control oocytes remained at the germinal vesicle stage. Immature cytoplasm did not induce nuclear maturation of recipient oocytes when injected. The cytoplasm of the oocytes injected with maturing cytoplasm preserved the capacity to cause GVBD in the second recipients. These results suggest that the level of injected maturation promoting factor (MPF) must rise before GVBD can occur. When the pig oocytes injected with 120 pl of mouse maturing cytoplasm were cultured in cycloheximide for 8 h, the presence of GVBD was dramatically reduced. Thus, in pig oocytes the increase in active MPF up to the effective concentration requires protein synthesis.

Animals↗
PubMed2002

Induction of maturation-promoting factor during Xenopus oocyte maturation uncouples Ca(2+) store depletion from store-operated Ca(2+) entry.

During oocyte maturation, eggs acquire the ability to generate specialized Ca(2+) signals in response to sperm entry. Such Ca(2+) signals are crucial for egg activation and the initiation of embryonic development. We examined the regulation during Xenopus oocyte maturation of store-operated Ca(2+) entry (SOCE), an important Ca(2+) influx pathway in oocytes and other nonexcitable cells. We have previously shown that SOCE inactivates during Xenopus oocyte meiosis. SOCE inactivation may be important in preventing premature egg activation. In this study, we investigated the correlation between SOCE inactivation and the Mos-mitogen-activated protein kinase (MAPK)-maturation-promoting factor (MPF) kinase cascade, which drives Xenopus oocyte maturation. SOCE inactivation at germinal vesicle breakdown coincides with an increase in the levels of MAPK and MPF. By differentially inducing Mos, MAPK, and MPF, we demonstrate that the activation of MPF is necessary for SOCE inactivation during oocyte maturation. In contrast, sustained high levels of Mos kinase and the MAPK cascade have no effect on SOCE activation. We further show that preactivated SOCE is not inactivated by MPF, suggesting that MPF does not block Ca(2+) influx through SOCE channels, but rather inhibits coupling between store depletion and SOCE activation.

Animals↗
PubMed2001

Maturation of marginal zone and follicular B cells requires B cell activating factor of the tumor necrosis factor family and is independent of B cell maturation antigen.

B cells undergo a complex series of maturation and selection steps in the bone marrow and spleen during differentiation into mature immune effector cells. The tumor necrosis factor (TNF) family member B cell activating factor of the TNF family (BAFF) (BLyS/TALL-1) plays an important role in B cell homeostasis. BAFF and its close homologue a proliferation-inducing ligand (APRIL) have both been shown to interact with at least two receptors, B cell maturation antigen (BCMA) and transmembrane activator and cyclophilin ligand interactor (TACI), however their relative contribution in transducing BAFF signals in vivo remains unclear. To functionally inactivate both BAFF and APRIL, mice transgenic for a soluble form of TACI were generated. They display a developmental block of B cell maturation in the periphery, leading to a severe depletion of marginal zone and follicular B2 B cells, but not of peritoneal B1 B cells. In contrast, mice transgenic for a soluble form of BCMA, which binds APRIL, have no detectable B cell phenotype. This demonstrates a crucial role for BAFF in B cell maturation and strongly suggests that it signals via a BCMA-independent pathway and in an APRIL-dispensable way.

Animals↗
PubMed1996

Delivery following intracytoplasmic injection of mature sperm cells recovered by testicular fine needle aspiration in a case of hypergonadotropic azoospermia due to maturation arrest.

This is the first reported delivery following intracytoplasmic sperm injection (ICSI) of mature live testicular sperm cells collected in a case of hypergonadotrophic azoospermia with maturation arrest. The 30 year old couple presented with primary infertility of 11 years duration, the man being submitted in childhood to five orchidopexy operations for the treatment of cryptorchism. He had elevated serum follicle stimulating hormone (FSH; 18.8 IU/I), an atrophic left testis and a normal sized right testis, the biopsy of which diagnosed maturation arrest and focal scarring. The couple refused donor insemination for religious reasons and the only option was an attempt at testicular sperm collection. Multiple testicular and epididymal fine needle aspirations were performed, using an aspiration handle loaded with 20 ml syringe and 21-23 gauge butterfly needles. The mature spermatozoa recovered were used to inseminate the oocytes by ICSI. Prior to this procedure, the patient's wife underwent ovulation induction using a long protocol of mid-luteal gonadotrophin-releasing hormone analogue/human menopausal gonadotrophin (GnRHa/HMG). At oocyte retrieval, ten oocytes were recovered. Eight live sperm cells were recovered from the aspirates of the right testis. Following ICSI into four metaphase II and two metaphase I oocytes, one mature oocyte was fertilized, cleaved and was transferred to the uterus 48 h after oocyte retrieval. The patient conceived and delivered a 3300 g boy at term. In conclusion, our results demonstrate that this novel approach should be considered in cases with hypergonadotrophic azoospermia due to testicular failure. Further experience is needed to establish the exact criteria for its use.

Adult↗
PubMed2001

The role of heat shock protein (hsp70) in dendritic cell maturation: hsp70 induces the maturation of immature dendritic cells but reduces DC differentiation from monocyte precursors.

Members of the heat shock protein (hsp70) family are either constitutively expressed (hsc70) or can be induced by hyperthermic stress (hsp70). Recombinant hsp70 (rhsp70) stimulates cytokine production from monocytes and enhances NK cell proliferation and cytotoxicity. Here we demonstrate that rhsp70 binds to immature dendritic cells (DC) derived from monocyte precursors and induces their maturation as evidenced by an increase in CD40, CD86 and CD83 expression. Immature DC stimulated to mature with rhsp70 show an enhanced ability to present tyrosinase peptide to specific CTL. Mature DC did not bind rhsp70, suggesting a down-regulation in the expression of its receptor. When rhsp70 was added to monocyte precursors at the same time as GM-CSF and IL-4 it reduced the differentiation of monocytes into DC as shown by a decrease in the level of CD40, CD83, CD86 and HLA-DR expression and an increase in CD14 expression. The constitutively expressed hsc70 had neither a stimulatory effect on the maturation of immature DC nor did it reduce the differentiation of monocytes into DC. These findings demonstrate the specific ability of rhsp70 to induce the maturation of immature DC. Therefore rhsp70 may be useful for its adjuvant like properties in DC based immunotherapy of certain tumors.

Antigens, CD↗
PubMed1998

GTH II but not GTH I induces final maturation and the development of maturational competence of oocytes of red seabream in vitro.

The effects of red seabream gonadotropins (PmGTH I and PmGTH II) on the induction in vitro of germinal vesicle breakdown (GVBD) and the development of maturational competence (responsiveness to maturation-inducing steroid) were examined in the oocytes of red seabream. PmGTH II was highly effective in inducing GVBD in both maturationally incompetent (45.6 +/- 3.2% GVBD at a concentration of 300 ng/ml) and competent oocytes (42.3 +/- 0.4% GVBD at a concentration of 300 ng/ml). 17,20beta-Dihydroxy-4-pregnen-3-one (DHP, 10 ng/ml) increased the frequency of GVBD induced by PmGTH II. PmGTH I (33, 100, 300, and 900 ng/ml) was unable to induce GVBD at any tested concentration in the presence or absence of DHP. Actinomycin D (1 microgram/ml) and cycloheximide (1 microgram/ml) totally inhibited the PmGTH II-induced GVBD in the presence and in the absence of DHP. Both PmGTH I and PmGTH II stimulated in vitro production of 11-ketotestosterone in sliced testes of red seabream in a similar potency. These results indicate that PmGTH II, but not PmGTH I, induces the final maturation of oocytes, as well as the development of the maturational competence of oocytes, in red seabream.

Animals↗
PubMed2003

Transient exposure of dendritic cells to maturation stimuli is sufficient to induce complete phenotypic maturation while preserving their capacity to respond to subsequent restimulation.

Dendritic cells (DC) are activated by pathogens, cytokines and activated T cells. We investigated the impact of a transient initial DC stimulation on the kinetics of maturation using a combination of double-stranded RNA and TNFalpha and subsequent restimulation by T cell-derived stimuli. Transient stimulation of DC was sufficient to start an irreversible program of phenotypic maturation which proceeded in the absence of the initial stimulus. Transiently stimulated DC secreted lower amounts of IL-12 during the 48-h period of the first stimulation than cells activated for 48 h. Although both DC preparations expressed the same level of maturation-associated markers at 48 h, DC stimulated for shorter periods preserved higher sensitivity to boosting upon subsequent stimulation by T cell-derived signals. We showed that DC initially stimulated for shorter periods were more potent stimulators of T lymphocytes and they induced a more polarized Th1 response. These results indicate that short exposure of DC to maturation stimuli enables an efficient defensive immune response induction by differentially regulating phenotypic maturation and cytokine production of DC.

Antigen Presentation↗
PubMed1985

Murine leukemia virus maturation: protease region required for conversion from "immature" to "mature" core form and for virus infectivity.

Murine leukemia virus (MuLV) genome encodes a protease (Y. Yoshinaka, I. Katoh, T.D. Copeland, and S. Oroszlan (1985), Proc. Natl. Acad. Sci. USA 82, 1618-1622), which has been shown to cause maturation, specified as morphological conversion from "immature" to "mature" form of virus cores. To examine whether "immature" particles have infectivity or not, we constructed mutant DNAs with deletions in the protease region. The NIH/3T3 cells transfected with mutant DNAs produced "immature" particles, having immature morphology and containing Pr65gag, a polyprotein precursor of core proteins. The specific infectivity of the extracellularly released and purified particles was shown to be greatly reduced based on reverse transcriptase activity and protein content as compared with the "mature" particles obtained from wild-type DNA-transfected cells. The mutant genomes encoded functionally normal surface glycoprotein, gp70. These results strongly suggest that maturation of MuLV from "immature" to "mature" form of virus particles is indispensable to virus infectivity. The importance of processing of gag and pol, as well as transmembrane protein precursors by the viral protease is discussed.

Amino Acid Sequence↗
PubMed1983

T-cell maturation in the human thymus and tonsil: peanut agglutinin binding T lymphocytes in thymus and tonsil differ in maturation stage.

The finding of peanut agglutinin (PNA) binding capacity, supposed to be a marker of immature lymphocytes, within the T-cell population of the human thymus (58%) and tonsil (10%) prompted the comparison of maturation stages of PNA binding (PNA+) and nonbinding (PNA-) T cells in both organs. The proliferative response after mitogenic stimulation of purified PNA+ fractions was significantly less than that of purified PNA- fractions. The results of mitogen dose-response experiments, of variation in time of culture harvest, and of addition of irradiated allogeneic peripheral blood non-T cells indicated the intrinsic mitogen unresponsiveness of cells in the PNA+ fractions. The mitogen response of tonsil fractions was higher than that of thymocyte fractions. Cells with an immature immunologic phenotype were enriched in the thymocyte PNA+ fraction, and almost absent in the tonsil fractions. Both tonsil fractions contained cells with the immunologic phenotype of mature T cells, and showed a purine interconversion enzyme makeup comparable to mature T lymphocytes. It is concluded that the tonsil PNA+ T cell is a functionally immature lymphocyte which is in a further maturation stage than PNA+ or PNA- thymocytes. The presence of PNA+ T cells outside the thymus is of relevance for the clinical evaluation of PNA binding assays and suggests the occurrence of T-cell maturation within the tonsil environment.

Cell Separation↗
PubMed1996

Appearance of neurofilament subunit epitopes correlates with electrophysiological maturation in cortical embryonic neurons cocultured with mature astrocytes.

E14 rat cortical neurons which have almost no glial progenitors were cocultured with a homogeneous population of mature type 1 astrocytes at a 4/1 ratio in serum free medium. Maturation of neurons was evaluated using a set of well characterized antibodies and two new monoclonal antibodies (MN2E4 and MN3H6) raised against various neurofilament subunits and whole-cell patch clamp experiments. We observed that this coculture method leads to a well-timed and very homogeneous neuronal maturation and that sequential appearance of neurofilament subunits in developing neurons correlates with the electrophysiological maturation. This sequence, early expression of the 68 kDa neurofilament subunit and late appearance of the 200 kDa neurofilament subunit, occurs in normal brain development, which validates this culture model as a useful tool for studying neuronal maturation and differentiation. MN2E4 staining (non-phosphorylated 200 kDa cytoskeletal protein antibody) appeared just before the neurons became excitable. It could thus be used as a functional neuronal marker. MN3H6 staining (phosphorylated 160-200 kDa neurofilament subunit antibody) appeared just after the neurons made synaptic contacts and generated synaptically driven spike bursts. This finding indicated that some phosphorylated epitopes of 160-200 kDa neurofilament followed synaptogenesis. These processes may play a key role in stabilizing the synapses to achieve a functional neuronal network.

Animals↗