Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Deciphering the complex series of regulatory events that occur during early development depends partly on the ability to accurately quantify stage-specific mRNA species. However, the paucity of biological material coupled with the lack of sensitivity and/or reproducibility of the currently available quantitative methods had been severe limitations on single cell analysis. Rapid cycle DNA amplification is a highly sensitive technique for amplification of specific DNA sequences. With the addition of fluorescence probes, it is possible to monitor the log-linear phase of amplification during which the most useful quantitative data is obtained. Unknown concentrations are extrapolated from standards co-amplified producing a standard curve. Furthermore, micro volume capabilities allow for the analysis of minute samples. Consequently, this approach is ideally suited to the needs of the clinical IVF laboratory. Rapid fluorescence monitored cycling was used to examine expression levels of the housekeeping genes beta-actin and hypoxanthine guanine phosphorlbosyltransferase in individual murine/human oocytes and/or embryos. Results obtained compared favourably with those attained by others and followed the predicted temporal patterns of expression. Once informative reproductive molecular markers are identified by micro-array analysis, minimally invasive techniques can be developed to biopsy cytoplasm and/or polar bodies for clinical evaluation using rapid fluorescence monitored reverse transcription-polymerase chain reaction methods.
Mol Hum Reprod 2000 May
PMID:Quantification of mRNA in single oocytes and embryos by real-time rapid cycle fluorescence monitored RT-PCR. 1077 49

Transgenic bovine sperm were produced by restriction enzyme mediated insertion (REMI). REMI utilizes lipofection of linearized pEGFP and the corresponding restriction enzyme for integration into the sperm genomic DNA. The transgenic sperm were used in IVF to produce morula expressing GFP. When transgenic sperm were used for AI in two cows, the resultant calves expressed the exogenous DNA in their lymphocytes as determined by (a) PCR and RT-PCR; (b) specific emission of green fluorescence by GFP; and (c) Southern blot analysis. Data demonstrate that REMI is an efficient method for the production of transgenic sperm and corresponding offspring by AI or embryos by IVF.
Mol Reprod Dev 2000 Jun
PMID:Gene integration into bovine sperm genome and its expression in transgenic offspring. 1082 91

We have recently found that values of the transforming growth factor (TGF)beta1 in human ovarian follicular fluid obtained during ovarian stimulation for IVF were higher in women who subsequently became pregnant following embryo transfer. We therefore postulated that TGFbeta1 may have a beneficial effect on the preimplantation embryo and improve the chances of a successful implantation. We have used reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry to investigate the presence in human oocytes and preimplantation embryos of the essential components of the TGFbeta signalling pathway, TGFbeta receptors type I and II and the substrate proteins Smad 2 and 3. We found that both receptors, as well as Smad 2 and 3, were present in the unfertilized oocyte, whereas only the type I receptor and Smad 2 and 3 were present at the blastocyst stage. At the 4-cell and 8-cell stages neither of the receptors was present, but Smad 2 and 3 were present at both stages. These findings support our hypothesis that the TGFbeta1 in follicular fluid may interact with the oocyte and preimplantation embryo via TGFbeta receptors, and that TGFbeta signalling may be important for the development of the oocyte and the preimplantation embryo.
Mol Hum Reprod 2000 Jun
PMID:TGFbeta receptor types I and II and the substrate proteins Smad 2 and 3 are present in human oocytes. 1082 65

In this study, we analysed the distribution of beta tubulins to detect spindle and cytoplasmic microtubules, alpha acetylated tubulins for sperm microtubules and chromatin configuration in oocytes showing fertilization failure after conventional IVF or intracytoplasmic sperm injection (ICSI). A total of 450 human oocytes that failed to fertilize were studied 20-40 h after IVF or ICSI. In all, 287 oocytes were stained for immunofluorescence and chromosomal spreads were performed by Tarkowski's air-drying method in 163 IVF or ICSI oocytes that did not develop pronuclei after the extrusion of a second polar body. Immunofluorescence analysis showed that the main reason of fertilization failure after IVF was no sperm penetration (55.5%). The remaining oocytes showed different abnormal patterns, e.g. oocyte activation failure (15.1%) and defects in pronuclei apposition (19.2%). On the other hand, fertilization failure after ICSI was mainly associated to incomplete oocyte activation (39.9%), and to a lesser extent with defects in pronuclei apposition (22.6%) and failure of sperm penetration (13.3%). A further 13.3% of the ICSI oocytes arrested their development at the metaphase of the first mitotic division. The chromosomal spreads allowed the analysis of abortive activations, in which no pronuclei formed but a second polar body was extruded. Immunofluorescence and cytogenetic analysis provided a useful tool to improve infertility diagnosis and prognosis in each particular case.
Mol Hum Reprod 2000 Jun
PMID:Cytoskeletal organization defects and abortive activation in human oocytes after IVF and ICSI failure. 1082 67

Advanced endometrial transformation often occurs in IVF and embryo transfer therapy after ovarian stimulation with gonadotrophins. One reason is probably the early rise in peripheral progesterone concentration after ovulation induction. Consequently, we studied in a rabbit model, whether the post-ovulatory application of the progesterone receptor antagonist, onapristone, could prevent such an advancement of endometrial transformation after stimulation with different gonadotrophin preparations. The inhibitory effect of onapristone on the endometrium is dependent upon the strength of ovarian stimulation. In unstimulated animals or animals treated with recombinant LH (nine corpora lutea/animal in both groups), secretory differentiation and proliferation of the endometrium was strongly inhibited by onapristone. After weak ovarian stimulation with a 3:1 mixture of FSH and LH (22 corpora lutea/animal), secretory differentiation was strongly inhibited, while proliferation was enhanced. After strong stimulation with either a 1:1 mixture of FSH and LH, or human menopausal gonadotrophin (HMG; >40 corpora lutea/animal), only limited inhibitory effects of onapristone on secretory transformation or proliferation could be detected. In conclusion, these graded effects of onapristone after stimulation with gonadotrophins, resemble the basic observations from which a therapeutic strategy emerges, to modulate the advanced endometrial transformation which occurs in many IVF patients after ovarian stimulation.
Mol Hum Reprod 2000 Aug
PMID:Modulation of endometrial transformation in gonadotrophin-stimulated and unstimulated pseudo-pregnant rabbits: studies with the progesterone receptor antagonist, onapristone. 1090 83

Adult animal cloning has progressed to allow the production of offspring cloned from adult cells, however many cloned calves die prenatally or shortly after birth. This study examined the expression of three important metabolic enzymes, lactate dehydrogenase (LDH), citrate synthase, and phosphofructokinase (PFK), to determine if their detection in nuclear transfer (NT) embryos mimics that determined for in vitro produced embryos. A day 40 nuclear transfer produced fetus derived from an adult cell line was collected and fetal fibroblast cultures were established and maintained. Reconstructed NT embryos were then produced from this cell line, and RT-PCR was used to evaluate mRNA reprogramming. All three mRNAs encoding these enzymes were detected in the regenerated fetal fibroblast cell line. Detection patterns were first determined for IVF produced embryos (1-cell, 2-cell, 6-8 cell, morula, and blastocyst stages) to compare with their detection in NT embryos. PFK has three subunits: PFK-L, PFK-M, and PFK-P. PFK-L and PFK-P were not detected in bovine oocytes. PFK subunits were not detected in 6-8 cell embryos but were detected in blastocysts. Results from NT embryo RT-PCR demonstrated that PFK was not detected in 8-cell NT embryos but was detected in NT blastocysts indicating that proper nuclear reprogramming had occurred. Citrate synthase was detected in oocytes and throughout development to the blastocyst stage in both bovine IVF and NT embryos. LDH-A and LDH-B were detected in bovine oocytes and in all stages of IVF and NT embryos examined up to the blastocyst stage. A third subunit, LDH-C was not detected at the blastocyst stage in IVF or NT embryos but was detected in all earlier stages and in mature oocytes. In addition, LDH-C mRNA was detected in gonad isolated from the NT and an in vivo produced control fetus. These results indicate that the three metabolic enzymes maintain normal expression patterns and therefore must be properly reprogrammed following nuclear transfer.
Mol Reprod Dev 2000 Aug
PMID:Genetic reprogramming of lactate dehydrogenase, citrate synthase, and phosphofructokinase mRNA in bovine nuclear transfer embryos produced using bovine fibroblast cell nuclei. 1091 95

A quality control (QC) system is needed in ART units to assure reproducibility of all methods and competence in all duties performed by the personnel. The necessity of a quality control system becomes even clearer when considering the possible risks of ART. It is therefore essential to have a system to assure that everybody knows exactly how everything should be done. Furthermore, a QC system should bring about improvements such as making activities and procedures clearer to the staff and making the working methods more flexible. QC was initially created for the industry, and has later been applied to other types of activities such as management of organizations, services like health care including different types of clinical testing laboratories. To maintain a high standard in our IVF laboratory, and to assure reproducibility of the methods used we decided to apply for accreditation according to the European Norm (EN) 45001 and requirements for the competence of testing laboratories ISO/IEC Guide 25. A process was started where all routines and methods within the laboratory were documented and finally the QC system was described in a quality manual. Application for accreditation was submitted to the Swedish board for accreditation and conformity assessment (SWEDAC). Our ART laboratory finally became accredited according to the EN 45001 and requirements for the competence of testing laboratories ISO/IEC Guide 25. Introducing and fully implementing a quality control system in our laboratory has standardized the methods and the way that the embryologists perform their work in the laboratory. It has also optimized the environment in which the patient's gametes and embryos are handled.
Mol Cell Endocrinol 2000 Aug 15
PMID:The application of quality systems in ART programs. 1098 1

There is a general consensus on the clinical fact that the more embryos replaced the higher pregnancy rates are achieved. For this reason those IVF cycles with a low response and a reduced number of oocytes and embryos will have very few chances of producing a pregnancy. It is very important to diagnose, by means of the anamnesis and hormonal tests which patients are most likely to present a poor response to conventional ovarian stimulation protocols. It is mandatory to know the patient's plasmatic levels of FSH and estradiol together with personal data such as the age and the previous history of the patient. Only young poor responders with a normal basal hormonal profile will have some chances that by applying new protocols and combining new drugs, improve their response and have higher pregnancy rates. For the old poor responders who have already failed to alternative protocols including natural cycles, oocyte donation is the last and best hope.
Mol Cell Endocrinol 2000 Aug 15
PMID:Stimulation protocols for poor responders and aged women. 1098 3

Men with non-obstructive azoospermia exhibit different histopathologic syndromes in the testicle biopsy, varying from aplasia, Sertoli cell only syndrome, maturation arrest and hypoplasia. The genetic basis of these syndromes is discussed. We present the diagnostic testicle biopsies performed on 160 consecutive non-obstructive azoospermic patients, and these results were correlated with the findings after multiple bilateral treatment testicle biopsy. Each syndrome had to be reevaluated as for the presence of at least one focus of spermatogenesis up to the primary spermatocyte, round spermatid, elongating spermatid, elongated spermatid, or spermatozoa stages. The clinical outcome using donor sperm-IVF, spermatid or sperm intracytoplasmic injection is thereafter presented. A new prognosis based on the findings of this large clinical series coupled to results obtained with Y chromosome molecular screening is offered. Alternative treatments to donor sperm for men without spermatids are discussed.
Mol Cell Endocrinol 2000 Aug 15
PMID:Prognostic factors for successful testicle spermatid recover. 1098 6

In previous studies we demonstrated that bovine cumulus oocyte complexes (COCs) obtained from small and medium sized follicles express growth hormone receptor (GHR) mRNA and respond to growth hormone (GH) addition during in vitro maturation. The aim of this study was to investigate whether bovine zygotes and preimplantation embryos continue the expression of GHR gene after in vitro fertilization and during early embryo development and whether supplementation of GH during embryo culture affects embryo development. Therefore, COCs obtained from small and medium sized follicles were cultured in M199 supplemented with 10% FCS and gonadotropins for 24 hr. After in vitro fertilization the embryos were cultured: (a) on a monolayer of buffalo rat liver (BRL) cells in M199 supplemented with 10% FCS and 100 ng/ml bovine GH (NIH-GH-B18); (b) in droplets of serum-free BRL-conditioned medium supplemented with 100 ng/ml GH; (c) in droplets of synthetic oviductal fluid (SOF) supplemented with 100 ng/ml GH. Cultures without GH served as controls. Embryos were scored morphologically and the efficiency of the culture system was evaluated (a) as the percentage of cleaved embryos 4 days after IVF, (b) the percentage of blastocysts on Day 9 expressed on the basis of the number of oocytes at the onset of culture, and (c) the percentage of hatched blastocysts on Day 11 expressed on the basis of the total number of blastocysts present at Day 9. For gene expression, immature (GV) and mature (MII) oocytes (as positive control), embryos with less than 8 cells, 16-32 cells, and hatched blastocysts were prepared for reverse transcriptase polymerase chain reaction (RT-PCR) to assess the expression of mRNA of GHR. Messenger RNA for GHR was found in GV and MII oocytes and in all stages of embryo development. No mRNA for GH could be detected in early and expanded blastocysts produced in SOF medium. Immunoreactive GHR was found both in trophoblastic and embryonic cells of hatched blastocysts. Addition of 100 ng/ml GH during embryo culture on a monolayer of BRL cells in M199 supplemented with 10% FCS did not affect embryo development. However, GH (100 ng/ml) supplementation during embryo culture in droplets of serum-free BRL conditioned medium significantly (P < 0.05) enhanced the proportion of > 8-cell embryos. Similarly, culture of embryos in droplets of SOF medium in the presence of GH (100 ng/ml) significantly (P < 0.05) enhanced the number of > 8-cell embryos from 53.8% in control to 70.6% in GH-treated group. Day 9 blastocyst formation in SOF medium was also significantly (P < 0.01) increased in the presence of GH (33.9%) compared to the control (20.2%). Embryos cultured in SOF without GH rarely resulted in hatched blastocysts (0.7%). However, GH supplementation remarkably enhanced the proportion of the hatched blastocysts (13%). In conclusion, expression of GHR gene in preimplantation bovine embryos, presence of the receptor, and the beneficial effect of GH on cleavage, blastocyst formation and hatchability of the embryos point to the involvement of GH in early embryonic development.
Mol Reprod Dev 2000 Nov
PMID:Preimplantation bovine embryos express mRNA of growth hormone receptor and respond to growth hormone addition during in vitro development. 1101 32


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