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Results for “In vitro maturation”

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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↗

PubMed2005

Effect of homologous follicular fluid from medium-sized and large follicles on in vitro maturation of equine cumulus-oocyte complexes.

The in vitro maturation (IVM) of equine oocytes is typically performed using various synthetic media; however, an optimal IVM system for equine oocytes has not been developed. The aim of the present study was to evaluate the effects of two types of follicular fluid (FF) obtained from cyclic mares and two incubation intervals for the IVM of equine cumulus-oocyte complexes (COCs). Follicular fluid was collected from medium-sized (20-29 mm diameter) and large (e30 mm; post-human chorionic gonadotrophin administration) follicles using transvaginal ultrasound-guided follicle aspiration. Compact (n = 232) and non-compact (n = 183) COCs obtained from a slaughterhouse were incubated separately in the following groups: (1) FF from medium follicles for 24 h; (2) FF from large follicles for 24 h; (3) control (synthetic) medium for 24 h; (4) FF from medium follicles for 24 h then FF from large follicles for an additional 24 h; (5) FF from large follicles for 48 h; and (6) control medium for 48 h. For compact COCs, there was a tendency (P = 0.06) for more COCs incubated in FF from large follicles for 24 h to reach metaphase II compared with those incubated in control medium for 24 h (58% v. 35%, respectively). More (P < 0.05) compact COCs had degenerated after incubation in control medium for 48 h compared with all other groups (51% v. 14-24%, respectively). For non-compact COCs, incubation in FF from medium follicles for 24 h resulted in more (P = 0.05) COCs at metaphase II compared with control medium for 48 h (58% v. 29%, respectively). These results indicate that homologous FF from cyclic mares is a suitable alternative for the IVM of equine COCs and that it may be superior to conventional media for longer (i.e. >24 h) incubation intervals.

Animals↗

PubMed2005

Relationship between equilibration times and the presence of cumulus cells, and effect of taxol treatment for vitrification of in vitro matured porcine oocytes.

The effects of the presence or absence of cumulus cells and equilibration times (1 and 4 min) with cryoprotectant, and Taxol treatment before vitrification (0.5-5.0 microM) on development of in vitro matured porcine oocytes after vitrification were examined. Ethylene glycol (30%) and sucrose (0.5M) was used as a vitrification solution (39 degrees C), and cryotop was used for cryo-container. There was a significant relationship (F value: 6.077, P<0.05) in the rate of morphologically normal oocytes after vitrification between the equilibration times and the presence or absence of cumulus cells. The blastocyst rates were not significantly different between Taxol (1.4-5.5%) and non-treated control (8.8%). The results show that the optimal exposure time to achieve survival after vitrification depends on the presence or absence of cumulus cells, and that Taxol has no positive effect on the developmental capacity of vitrified, in vitro matured porcine oocytes.

Animals↗

PubMed1991

In-vitro matured human macrophages express Alzheimer's beta A4-amyloid precursor protein indicating synthesis in microglial cells.

Microglia which are consistently associated with Alzheimer's disease (AD) senile plaques are part of the mononuclear phagocyte system. In-vitro matured human monocyte-derived macrophages feature many immunological characteristics of microglia. We found strong constitutive expression of Alzheimer's beta A4-amyloid precursor protein (APP) in human mononuclear phagocytes after terminal in-vitro maturation from monocytes to macrophages. Amyloid has previously been found to be associated with microglia in AD brains, however, it remained unclear whether the material was synthesized in or had been phagocytosed by the cells. The findings presented here support the assumption that brain microglia may contribute to APP synthesis in AD brain.

Amyloid beta-Peptides↗

PubMed1998

Pretreatment with follicle stimulating hormone promotes the numbers of human oocytes reaching metaphase II by in-vitro maturation.

The objective of this study was to investigate the effect of follicle stimulating hormone (FSH) priming on the in-vitro maturation (IVM) of human oocytes from healthy ovaries using a chemically defined culture system. Seventeen patients donating oocytes for research received a truncated course of 600 IU FSH over 5 days and a further control group of nine patients received no FSH treatment. Mid-follicular phase cumulus-enclosed oocytes (n = 160) were aspirated from follicles < or =4 mm diameter under transvaginal ultrasound guidance and were cultured for 48 h in microdrops of medium containing 10 mIU/ml FSH and 100 mIU/ ml human chorionic gonadotrophin (HCG). The results demonstrated that human oocytes will efficiently undergo IVM under serum-free conditions. After mild FSH stimulation, a greater number of cumulus-enclosed oocytes was collected, and following culture, a lower rate of degeneration was observed. Significantly more oocytes completed nuclear maturation to metaphase II following FSH stimulation (71.1 versus 43.5%). In conclusion, a truncated course of FSH stimulation in vivo improved the oocyte maturation rate in vitro, giving a mean of 4.8+/-0.7 metaphase II oocytes per patient compared with only 2.1+/-0.7 from control patients, thus yielding more mature oocytes for future IVF treatment.

Adult↗

PubMed2006

Cryopreservation of in vitro matured buffalo (Bubalus bubalis) oocytes by minimum volumes vitrification methods.

The aim of this study was to evaluate the efficiency of the solid surface vitrification (SSV) and the cryoloop vitrification (CLV) methods to cryopreserve in vitro matured buffalo oocytes. Another objective of the work was to investigate whether the presence of cumulus cells affects the efficiency of oocyte vitrification in this species. In the SSV method, oocytes were vitrified in a solution of 35% ethylene glycol, 5% polyvinyl-pyrrolidone and 0.4% trehalose and they were warmed in a 0.3M trehalose solution. In the CLV method, oocytes were vitrified in 16.5% ethylene glycol and 16.5% dimethyl sulfoxide and warmed in decreasing concentrations of sucrose. The oocytes that survived vitrification were fertilized and cultured in vitro up to the blastocyst stage. Although high survival rates were recorded in all groups, when the oocytes were vitrified by the CLV method in the absence of cumulus cells, the survival rate was significantly (P<0.05) lower. However, the CLV gave a significantly higher cleavage rate compared to the SSV with the denuded oocytes (45% versus 26%, respectively; P<0.05), whereas no differences were found between methods with the cumulus-enclosed oocytes (14% versus 15%, respectively). Blastocysts were produced for the first time from in vitro matured oocytes that were vitrified-warmed in buffalo. Nevertheless, vitrification significantly decreased blastocyst yield, regardless of both the method employed and the presence or absence of cumulus cells.

Animals↗

PubMed2004

In-vitro maturation of germinal-vesicle oocytes and cryopreservation in metaphase I/II: a possible additional option to preserve fertility during ovarian tissue cryopreservation.

This study describes the possibility of combining two options in order to preserve female fertility: cryopreservation of human ovarian tissue and in-vitro matured germinal vesicle (GV) oocytes retrieved during tissue dissection. In contrast to ovarian tissue cryopreservation, the cryostorage of in-vitro matured unfertilized metaphaseI/II oocytes could be a more realistic option. This concept of preserving fertility before chemotherapy and/or radiotherapy without a long time delay could be an additional reason for favouring ovarian tissue cryopreservation. This concept is discussed in regard to two cases.

Adult↗

PubMed1997

Stimulatory effect of growth hormone on in vitro maturation of bovine oocytes is exerted through the cyclic adenosine 3',5'-monophosphate signaling pathway.

The aim of this study was to investigate whether the stimulatory effect of growth hormone (GH) on the in vitro maturation and cumulus expansion of bovine oocytes is exerted through the cAMP or the tyrosine kinase pathway. Therefore bovine cumulus-oocyte complexes (COCs) were cultured in Medium 199 without fetal calf serum and gonadotropins, but supplemented with 100 ng/ml bovine GH (bGH; NIH-GH-B18) with or without 10 microM methyl 2,5-dihydroxycinnamate (erbstatin analogue), a specific tyrosine kinase inhibitor; 100 microM 2',3'-dideoxyadenosine (DDA), a specific adenylate cyclase inhibitor; or 10 microM H-89, a specific inhibitor of cAMP-dependent protein kinase A. Epidermal growth factor (EGF; 20 ng/ml) was added as a positive control for tyrosine kinase activation, and FSH (0.05 IU/ml) was added as a positive control for cAMP mediation during in vitro maturation in the absence or presence of the inhibitors. Culture was performed at 39 degrees C in a humidified atmosphere with 5% CO2 in air. To assess the effect on nuclear maturation, the proportion of oocytes in metaphase II stage after 16 h of culture was determined using 4,6-diamino-2-phenylindole staining. To determine the effect on cumulus expansion, the diameter of COCs at the onset and after 24 h of culture was measured. The stimulatory effects of GH on oocyte maturation and cumulus expansion were blocked by DDA and H-89 (p < 0.01). Similarly, FSH-induced cumulus expansion was abolished by DDA and H-89 (p < 0.05), while DDA did not block either EGF-induced oocyte maturation or cumulus expansion. Erbstatin analogue significantly blocked the stimulation of oocyte maturation and cumulus expansion by EGF (p < 0.02) but did not inhibit GH action on the COCs. It is concluded that the stimulatory effect of GH on oocyte maturation and cumulus expansion is mediated by the cAMP signal transduction pathway and not by JAK2 phosphorylation.

Adenylyl Cyclase Inhibitors↗

PubMed1999

[Oocyte of domestic mammals: a model for the study of in vitro maturation].

Oocyte maturation represents the final step of a long differentiation process that allows this very special cell to fully express its reproductive task. During maturation the oocyte nucleus, blocked at the late prophase of meiosis from the end of foetal life, resumes meiosis and progress to the metaphase II stage. Beyond these nuclear aspects, oocyte maturation also involves cytoplasmic modifications including well known morphological progression as well as poorly understood biochemical changes that are determinant for successful fertilisation and early embryo development. In physiological conditions, maturation occurs in the preovulatory follicle after the ovulatory surge of gonadotropins, in a complex and changing environment. This complexity leaded to the formulation of the first in vitro maturation systems involving tissue culture media supplemented with biological fluids. A more precise study of the effect of individual medium components allowed the design of more simple maturation conditions providing more reproducible results with less sanitary risks. Amongst maturation activating factors, the epidermal growth factor (EGF) seems to play a key function in several species. Other factors such as hormones, ovarian peptides (inhibin, activin) and other growth factors may also be involved but the interplay between these factors remains to be clearly established. Improvement of in vitro maturation techniques allowed to evaluate the importance of oocyte intrafollicular differentiation before maturation on the resulting developmental competence. The increased knowledge of the regulation of intrafollicular meiotic arrest now allow the design of a prematuration step to allow oocytes from smaller follicles to complete their differentiation in vitro. This improvement will allow a larger use of the huge reproductive potential stored in the ovary.

Animals↗

PubMed2000

[Future perspectives: oocyte cryopreservation and in vitro maturation].

After treatment for neoplasia, a young patient can nowadays hope to be pregnant. The aim of this paper is to establish a review of the literature about cryopreservation of oocytes and ovarian tissue, and in vitro maturation of oocytes. These studies also concern patients for which ovarian stimulation is difficult, such as PCOS. These techniques could also facilitate the creation of an oocyte bank as the one already existing for sperm. So far the main studies published, in spite of very timid first results, allow us to look to a great future for cryopreservation and in vitro maturation of oocytes.

Cryopreservation↗

PubMed1996

In vitro fertilization of in vitro matured oocytes obtained from the follicles without hCG exposure for prevention of severe ovarian hyperstimulation syndrome: a case report.

We aspirated ovarian follicles of an infertile patient who encountered a great risk of ovarian hyperstimulation (OHSS) during her ovulation-induction cycle, which had been stimulated with gonadotropins, in order to avoid OHSS without interrupting the treatment. If this action were to yield a considerable number of immature oocytes, some of which were to achieve in vitro maturation and subsequent fertilization, this method could be an alternative to conventional ovulation-induction methods for patients who repeatedly present imminent signs of hyperstimulation. However, the number of oocytes collected from the patient was disappointingly low, even though a large number of follicles were thoroughly aspirated. One of these oocytes was matured in vitro in a medium containing 30% follicular fluid and granulosa cells of mature follicles, fertilized, cryopreserved, and transferred in a later artificial cycle; but pregnancy was not achieved. A low number of recovered oocytes from patients in this situation might be a factor that greatly limits the clinical applications of this method.

Adult↗

PubMed2006

In vitro maturation of human oocytes.

Immature oocyte retrieval followed by in vitro maturation (IVM) opens a new horizon for modern assisted reproductive technologies (ART). Recent studies in IVM make it a feasible alternative to in vitro fertilization. Antral follicle count is correlated with the pregnancy rate, so women with polycystic ovarian syndrome or polycystic ovaries are the best candidates for IVM. IVM can also be offered to women at risk of ovarian hyperstimulation syndrome or poor responders. From the available information, IVM is a safe procedure and does not increase congenital anomalies or pregnancy complications. Further research is necessary in order to apply this technique to other patients.

Cells, Cultured↗

PubMed1999

Intrafollicular content of luteinizing hormone receptor, alpha-inhibin, and aromatase in relation to follicular growth, estrous cycle stage, and oocyte competence for in vitro maturation in the mare.

The intrafollicular content of LH receptor, alpha-inhibin, and aromatase are known good indicators of follicular status. We investigated the amounts of these proteins in granulosa and cumulus cells in relation to oocyte competence for in vitro maturation, follicular growth, and estrous cycle stage in the mare. Follicular punctures were performed 34 h after an injection of crude equine gonadotropins, either during the follicular phase, at the end of the follicular phase, or during the luteal phase. The cumulus-oocyte complex, granulosa cells, and follicular fluid of follicles larger than 5 mm were collected. The nuclear stage of the oocytes after in vitro culture was determined microscopically. Granulosa and cumulus cell amounts of LH receptor, alpha-inhibin, and aromatase were assessed by the semiquantitative Western blot method and image analysis. Follicular fluids were assayed for progesterone (P4) and estradiol-17beta (E2). The three factors were expressed in mural granulosa and cumulus cells from all follicles from the gonadotropin-independent growth period until the preovulatory stage. Considering all the follicles punctured, the amounts of LH receptor and alpha-inhibin in granulosa cells were not different for the three physiological stages studied. The amounts of aromatase in granulosa cells, as well as the E2:P4 ratios, were higher for follicles punctured during the follicular phase than for the two other groups (p < 0.05). Considering the data from the three groups, the E2:P4 ratio and the LH receptor and aromatase contents, but not alpha-inhibin, in granulosa cells increased with an increase in follicular diameter (p < 0.01). The E2:P4 ratios and the amounts of LH receptor, alpha-inhibin, and aromatase in granulosa cells were lower in follicles 5-9 mm in diameter than in larger ones (p < 0.05). In cumulus cells, the amounts of the three factors were different neither between the three groups nor between the follicular diameters. Although we could not establish any obvious relationship to oocyte competence for in vitro maturation, the influence of the follicle diameter on the content of LH receptors, alpha-inhibin, and aromatase in granulosa cells was similar to the influence of follicle diameter on oocyte competence. Therefore, one can hypothesize that, in the mare, there is a link between the acquisition of oocyte competence and the expression of these factors in the follicular cells.

Animals↗

PubMed1997

Quality of frozen-thawed testicular sperm and its preclinical use for intracytoplasmic sperm injection into in vitro-matured germinal-vesicle stage oocytes.

OBJECTIVE: To study the effects of cryopreservation on the quality of human testicular spermatozoa and the efficiency of intracytoplasmic sperm injection (ICSI) with frozen-thawed testicular sperm into metaphase II oocytes in vitro-matured from the germinal-vesicle stage oocyte. DESIGN: Preclinical freezing study on supernumarary testicular spermatozoa after ICSI. SETTING: Tertiary IVF center coupled with an institutional research environment. PATIENT(S): Twenty-nine patients undergoing excisional testicular biopsy for ICSI. INTERVENTION(S): Isolated testicular spermatozoa were cryopreserved and thawed; frozen-thawed motile testicular spermatozoa were microinjected. MAIN OUTCOME MEASURE(S): Prefreezing and post-thawing motility and viability, survival rate, fertilization rate, cleavage rate, and embryo quality after ICSI. RESULT(S): Mean percentage motility decreased from 21% before freezing to 6% after thawing. Vitality was impaired to a similar extent, decreasing from 68% to 22% (32% recovery rate). Injection of frozen-thawed testicular spermatozoa into in vitro-matured oocytes resulted in a fertilization rate of 50.9%. Cleavage rate was severely impaired. Half of the fertilized oocytes became arrested in the one-cell stage. CONCLUSION(S): Despite the low quality of the fresh testicular spermatozoa, a high percentage of prepared testicular sperm fractions showed survival and motility after the freezing and thawing process. Injection of frozen-thawed testicular sperm into matured oocytes resulted in fertilization rates comparable with these with fresh testicular sperm, but cleavage rates were severely impaired, which might be due to source of oocytes used for ICSI.

Adult↗

PubMed2005

Duration of in vitro maturation of recipient oocytes affects blastocyst development of cloned porcine embryos.

This study investigated the effects of different incubation periods for oocyte maturation and contact inhibition of donor cells as well as different osmolarities for storage of recipient oocytes on fusion rates, cleavage rates, and blastocyst yields of porcine somatic nuclear transfer (SCNT) derived embryos. In addition, the in vivo developmental potential of cloned embryos derived from the most promising SCNT protocol was tested by transfer to recipient gilts. Storage of in vitro-matured oocytes for 7.5 h in calcium-free TL-HEPES medium at 295 or 320 mOsmol prior to activation yielded significantly (p < 0.05) higher parthenogenetic blastocyst rates compared to storage in TL-HEPES with an osmolarity of 270 mOsmol (24.4 +/- 3.0% and 26.2 +/- 4.3% vs. 18.3 +/- 6.4%, respectively, mean +/- SD) and improved the visibility of the polar body. Electrical fusion of fibroblasts to enucleated oocytes matured for 38, 40, or 42 h resulted in similar fusion and cleavage rates (74.8-84.4%). However, nuclear transfer with oocytes matured for 40 h in vitro yielded significantly higher (p < 0.05) development to the blastocyst stage after 7 days of culture (14.7 +/- 1.7%) than with oocytes matured for 38 h (9.5 +/- 2.1%) or 42 h (5.1 +/- 2.1%). Contact inhibition for 24, 48, or 72 h significantly (p < 0.05) increased the proportion of cells at G0/G1 compared with cycling fibroblasts. However, duration of contact inhibition of the donor cells for either 24, 48, or 72 h had no effect on blastocyst rates of SCNT embryos. Four gilts received an average of 150 SCNT embryos (range 138-161) reconstructed with oocytes matured for 40 h; two of these became pregnant; one of them went to term and farrowed four piglets on day 115 of pregnancy. Microsatellite analysis confirmed that the clones were genetically identical with the donor cells. These results show that changes of the in vitro maturation protocol may affect in vitro development of reconstructed porcine embryos, while duration of the contact inhibition period plays a minor role for the success of porcine SCNT. The effects on in vivo development are yet to be determined.

Animals↗

PubMed1986

Characterization of small and large human peripheral blood monocytes: effects of in vitro maturation on hydrogen peroxide release and on the response to macrophage activators.

Human peripheral blood monocytes (HPBM) isolated from normal donors by centrifugal elutriation were divided into two populations according to volume. (Median volumes of small monocytes (SM) and large monocytes (LM) were 255 micron and 280 micron, respectively.) H2O2 production was determined during in vitro culture and in response to bacterial lipopolysaccharide (LPS), and to recombinant human interferon-gamma (rIFN-gamma). On day 1, H2O2 production by LM was significantly greater than that by SM. In vitro culture of SM resulted in an augmented ability to produce H2O2. By day 3, SM were the major H2O2 producers. Freshly isolated SM and LM, exposed for 24 hr to LPS and rIFN-gamma, showed different patterns of activation. Both SM and LM responded to LPS, with LM responding maximally at lower doses than SM. Only SM showed a significant augmentation of H2O2 production with rIFN-gamma treatment. We also assessed the effect of in vitro culture with activation. SM but not LM showed an increased H2O2 to LPS and rIFN-gamma after 7 days in culture. Continuous exposure of SM to rIFN-gamma resulted in maximal H2O2 production at day 3 of culture; this pattern was not seen for LPS. The production of H2O2 by HPBM is related to in vitro maturation. The enhanced H2O2 production by HPBM upon exposure to rIFN-gamma may be related to the induction of in vitro maturation.

Cell Differentiation↗

PubMed1993

Expression and modulation of a mononuclear phagocyte differentiation antigen (PAM-1) during in vitro maturation of peripheral blood monocytes.

Human macrophages obtained by in vitro maturation of peripheral blood monocytes express a surface antigen, PAM-1, recognized by a monoclonal antibody and typical of pulmonary alveolar and tissue macrophages. PAM-1, undetectable in freshly isolated peripheral blood monocytes, was expressed in monocyte-derived macrophages after 3 days of in vitro adherent culture and was maximal after 14-15 days (50%-60% of positive cells). Similar levels of PAM-1 positivity were observed in non-adherent monocyte-derived macrophages suggesting that cell adhesion was not a critical requisite for the expression of this antigen. Bacterial lipopolysaccharide and a monocyte chemotactic protein preparation respectively suppressed and upregulated PAM-1 expression in monocyte-derived macrophages. In contrast, interferon-gamma, although enhancing the levels of class II HLA-DR antigen in monocyte-derived macrophages, did not influence the kinetics of appearance and the levels of PAM-1 in these cells. Thus, expression of PAM-1, which is restricted to certain stages of the monocyte-macrophage differentiation pathway, is also differentially modulated by activation signals, which can be present in the micro-environment of inflammed tissues.

Antigens, Differentiation↗

PubMed2004

In vitro maturation and fertilization of oocytes from an intact ovary of a surgically treated patient with endometrial carcinoma: case report.

Polycystic ovary syndrome (PCOS) with prolonged anovulation had resulted in endometrial carcinoma in a 43-year-old woman. Since she and her husband did not share common biological children, they requested fertility preservation. Due to the woman's age, high dose progesterone and postponing surgery were both considered inappropriate. We therefore proposed oocyte retrieval from the ovaries removed by staging laparotomy followed by in vitro maturation and ICSI. Surrogacy could then enable a future pregnancy. Fourteen of 17 (82%) retrieved oocytes matured in vitro. Following ICSI, eight embryos (two at the pronuclear stage and six cleaved) were cryopreserved. To the best of our knowledge, this is the first report of oocyte aspiration, maturation and fertilization from an ovary removed by laparotomy.

Adult↗