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Query: UNIPROT:P06889 (Mol)
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Efforts to improve bovine embryonic development in vitro involved study of effects of thyroid stimulating hormone (TSH) alone or in combination with LH on bovine oocyte maturation (IVM). Putative effects were assessed by observing cumulus expansion (CE), fertilization (IVF), and development to morulae/blastocysts (M/B). Effects of prolactin (PRL) were also investigated. Variables for the 24-hr IVM interval were no hormone (control), TSH (0.1, 0.5, or 1.0 micrograms/ml) or PRL (10, 100, or 1000 micrograms/ml), luteinizing hormone (LH) (0, 10, or 100 micrograms/ml) + TSH (0.1 or 0.5 micrograms/ml), and serum (20%, v/v) + 0.5 micrograms TSH/ml; data were from 4-5 trials for each IVM treatment. Higher proportions of oocytes exhibited complete CE with hormones or serum than without (P less than 0.05). All oocytes (with and without CE) were inseminated with heparin-capacitated sperm. A higher proportion of inseminated oocytes cleaved after IVM with 0.5 micrograms TSH/ml (53.4%) than for other TSH treatments (P less than 0.05). The combination of TSH (0.1 and 0.5 micrograms/ml) with 10 micrograms LH/ml for IVM enabled higher proportions (P less than 0.05) of ova to fertilize (67.4 and 69.2%) than did medium alone (28.3%), LH (10 micrograms/ml) alone (54.1%) or serum + 0.5 micrograms TSH/ml (55.6%). No improvement in proportions undergoing fertilization was seen after addition of TSH to 100 micrograms LH/ml for IVM. Frequency of CE and cleavage did not differ among PRL treatments. More M/B developed from cleaved ova after IVM with LH or TSH than with PRL or no hormone (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Reprod Dev 1992 Feb
PMID:Thyroid stimulating hormone enhancement of bovine oocyte maturation in vitro. 159 83

The rate of cleavage and the onset of embryonic transcription of bovine embryos cultured in vitro (IVC) has been investigated. Embryos were derived from in vitro matured, in vitro fertilized oocytes (IVM/IVF) to improve developmental synchrony. The rate of cleavage was assessed by morphological evaluation between the one- and eight- to 16-cell stage. The rate of cleavage was found to be equivalent to that reported for in vivo recovered embryos. To assess the onset of embryonic transcription, embryos were cultured to the eight- to 16-cell stage in the presence of alpha-amanitin for various periods of time followed by two-dimensional polyacrylamide gel electrophoresis. Embryos readily cleaved to the eight- to 16-cell stage in the presence of inhibitor. alpha-Amanitin-sensitive protein synthesis was first detected at 36-48 h post-insemination (hpi) and continued up to 84 hpi. We conclude that bovine embryos produced by IVM/IVF/IVC are competent to initiate embryonic transcription at 36-48 h post-insemination and suggest that in vitro-induced cleavage arrest is not due to failure of the embryonic genome to initiate transcription.
Mol Reprod Dev 1991 Jun
PMID:Embryonic transcription in in vitro cultured bovine embryos. 187 21

The consequences of interactions between porcine sperm, eggs, and oviduct cells before and during fertilization in vitro (IVF) has been examined with particular reference to the block to polyspermy. The pattern of polypeptides secreted by porcine oviduct epithelial cells has been determined and its effects on sperm both during pre-fertilization co-culture and during fertilization have been examined. In standard IVF procedures with no oviduct cell involvement, high rates of penetration (91%) were accompanied by equally high rates of multiple sperm penetration (91% of penetrated eggs). Fertilization on oviduct cell monolayers or a combination of 1 h co-culture of sperm and oviduct cells before the addition of in vitro matured oocytes did not reduce polyspermy. However, a sperm-oviduct cell co-culture period of 2.5 h followed by IVF on oviduct cells selectively reduced the rate of polyspermy by 40% and 50% in two separate series of trials (United Kingdom and Japan, respectively): Overall fertilization rates after this treatment were high (95% or 84%, respectively). A 3.5 h period of pre-fertilization co-culture further reduced polyspermy to only 14% of penetrated eggs, but this treatment was accompanied by a sharp drop in the fertilization rate from an overall mean of 88% for all other groups to 19% after 3.5 h co-culture.
Mol Reprod Dev 1990 Aug
PMID:Effect of oviduct cells on the incidence of polyspermy in pig eggs fertilized in vitro. 222 87

In a previous study, we have shown that the cryopreservation of mouse oocytes caused increases in the rates of degeneration and of digynic polyploid embryos, while the fertility of frozen-thawed oocytes was decreased. In this study, we have attempted to determine the different stages in the complete freezing-thawing process which are deleterious for the oocytes and the subsequent zygotes. IVF assays showed that DMSO decreased the fertility of oocytes, whereas cooling to 0 degrees C had no effect. DMSO, used at 0 degrees C, was less deleterious for oocytes. Thus, the prefreezing manipulations seem to be important for the quality and fertility of oocytes. However, neither DMSO nor cooling increased the incidence of chromosomal abnormalities in embryos obtained from inseminated exposed oocytes. Therefore, the increased frequency of polyploidy observed in embryos after the cryopreservation of mouse oocytes must correspond to disruption occurring during the freezing-thawing process.
Mol Reprod Dev 1995 Jan
PMID:Effects of cooling and equilibration in DMSO, and cryopreservation of mouse oocytes, on the rates of in vitro fertilization, development, and chromosomal abnormalities. 770 64

The ability of human follicular fluid (hFF), retrieved from women undergoing IVF to induce the acrosome reaction (AR) in human sperm has been documented by several laboratories. However, the nature of the active factors in the hFF and the physiological meaning of the AR induction are highly controversial. We performed a three step purification scheme for hFF and all the fractions were screened for the AR-inducing activity. AR activity was associated with a protein fraction of M(r) > 180 kD that on further analysis under PAGE was found to be composed by subunits of apparent M(r) 50,000 and 29,000. The N-terminal sequences of these bands showed a 100% homology with the heavy and light chains of human IgG. A polyclonal antibody raised against the purified protein and anti-human IgG were both able to suppress the acrosome reaction-inducing activity of crude hFF. However, neither normal human serum nor a purified preparation of human IgG were able to mimic the AR-inducing activity of hFF. We concluded that the AR-inducing activity of hFF is, at least in part, due to the presence of antisperm antibodies.
Mol Reprod Dev 1994 Nov
PMID:Immunoglobulins from human follicular fluid induce the acrosome reaction in human sperm. 788 67

Development of 8-cell bovine embryos derived from in vitro matured/in vitro fertilized (IVM/IVF) oocytes was evaluated in two simple, serum-free media (CZB and SOM) with buffalo rat liver cells co-culture (BRLC) or after conditioning compared to a commonly used, serum-supplemented complex medium TCM-199. In a 3 x 4 factorial design, 578 eight-cell embryos were randomly assigned to 12 treatment groups. The factors were: first, type of culture medium (M199/FBS, CZBg and SOM), and second, the use of BRLC (as co-culture or to condition media for 24 hr and 48 hr) and unconditioned media. Development to morula was not affected by the type of medium, but co-culture and 48 hr conditioning within media type resulted in better development when compared to the 24-hr conditioned or unconditioned groups. Blastocyst development in SOM (38.9%) was different (P < 0.05) than in CZBg (46.6%) and M199/FBS (48.7%) and was lowest in the unconditioned group (27.8%) followed by 24 hr conditioned (33.3%), 48 hr (56.3%), and co-culture (59.6%). No blastocyst expansion was observed with unconditioned media and 24 hr conditioned SOM. Significant differences (P < 0.05) were found among all treatment groups except the co-culture and 48-hr conditioned groups. Hatching occurred only with co-culture and 48-hr conditioned groups of M199/FBS and CZBg media. These data show that CZB with glucose conditioned by BRLC monolayers for 48 hr can support the development of IVM/IVF produced bovine embryos to blastocyst compared to culture in TCM-199 with serum.
Mol Reprod Dev 1994 Jul
PMID:Development of IVM-IVF produced 8-cell bovine embryos in simple, serum-free media after conditioning or co-culture with buffalo rat liver cells. 791 75

The aim of the present series of experiments was to investigate the effect of the size of follicle from which the oocytes originate on their subsequent in vitro developmental ability. Ovarian follicles were isolated and grouped according to size (2-6 mm, > 6 mm). Primary oocytes were carefully liberated and grouped according to morphology into one of five categories: denuded; expanded; with two or three layers of cumulus; with four or five layers; and with many (six or more) layers. Following in vitro maturation (IVM), fertilization (IVF), and culture (IVC), more oocytes with many layers of cumulus (P < 0.01, 70.2%, 73/104 vs. 46.8%, 87/186, respectively) and a higher proportion of blastocysts were obtained from follicles > 6 mm compared to 2-6 mm follicles (P < 0.01, 65.9%, 60/91 from > 6 mm follicles vs. 34.3%, 34/99 from 2-6 mm follicles, respectively). Use of follicular fluid (BFF) from follicles of different sizes in the IVM medium did not significantly increase the cleavage rate or blastocyst yield compared to controls. Administration of porcine follicle-stimulating hormone (pFSH) to donors prior to slaughter was investigated as a possible means of increasing the number of larger sized follicles in the ovaries and, thereby, the quality of the recovered oocytes. It was found that administration of six injections of pFSH beginning 3 days prior to slaughter resulted in a significant increase (P < 0.001) in the proportion of follicles > 6 mm in diameter (31.6%) compared to that in nontreated controls (6.6%) and to animals that received only four injection groups (9.4%).(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Reprod Dev 1994 Jan
PMID:Effect of follicle size on bovine oocyte quality and developmental competence following maturation, fertilization, and culture in vitro. 812 30

Chromatin and microtubule configurations during the first cell cycle of bovine zygotes were analyzed by DNA staining and microtubule immunolocalization using an IVM/IVF system and oocytes matured and fertilized in vivo, in order to investigate the origin of the active centrosome and to characterize the nuclear and the cytoplasmic changes following bovine fertilization. Our results suggest that the paternal centrosome is active during early zygotic development, forming a conspicuous sperm aster soon after fertilization. We also report that polyspermy in bovine eggs, leads to the formation of numerous sperm asters with different degrees of association with the chromatin. The maternal structures in both monospermic and polyspermic zygotes can be lost or degenerate. Consequently, these cells may resume the first cell cycle as androgenotes, very often with several types of mitotic activity taking place in different regions of the cell cytoplasm at the same time. As indicated by a comparison of monospermic and polyspermic fertilization rates to rates of development, it is possible that some androgenetic embryos cleave and develop to the blastocyst stage.
Mol Reprod Dev 1993 Sep
PMID:Chromatin and microtubule morphology during the first cell cycle in bovine zygotes. 839 27

Various procedures have been reported for successful in vitro maturation and in vitro fertilization (IVM/IVF) of bovine follicular oocytes. Direct comparisons of these different recommended procedures have been rare. In this research, involving a total of 5,128 oocytes, a series of experiments were conducted to compare oocyte maturation, fertilization, and development in vitro with 2 maturation systems (with or without added hormones) and 3 types of sperm treatment procedures. Oocytes were collected from ovarian antral follicles (2-7 mm in diameter) within 3 hr after slaughter of cows or heifers. Those with intact or at least 4 layers of cumulus cells were selected for IVM/IVF. Oocytes were incubated for 22 hr in either Medium 199 with 7.5% fetal calf serum (M199 + FCS) alone or M199 + FCS with added hormones (M199 + FCS + H; oFSH 0.5 micrograms/ml, oLH 5.0 micrograms/ml, and E2 1.0 micrograms/ml) at 39 degrees C in 5% CO2 and 95% air. For IVF, frozen-thawed sperm were treated with either 0.1 microM calcium ionophore A23187 (A23187) for 1 min, or 10 or 100 micrograms/ml heparin (H10 or H100) for 15 min.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Reprod Dev 1993 Jan
PMID:Bovine oocyte development following different oocyte maturation and sperm capacitation procedures. 841 24

There are many factors affecting the efficiency of nuclear transfer technology. Some are evaluated here using our novel approach by enucleating oocytes at 20-22 hr after in vitro maturation (IVM), culturing the enucleated oocytes (cytoplasts) for 8-10 hr or 18-20 hr to gain activation competence and then conducting nuclear transfer. In the first experiment, we demonstrated that cumulus cell (CC) monolayer can support some cloned embryos to develop into morulae or blastocysts. Co-culture with CC and bovine oviduct epithelial cell (BOEC) monolayers resulted in no differences (P > 0.05) in supporting the development of cloned embryos (Experiment 2). When in vitro matured oocytes were enucleated at 22 hr after IVM followed by nuclear transfer 18-20 hr later, cleavage and morula or blastocyst development of the cloned embryos were similar to those resulting from the enucleated oocytes which had been matured in vivo (Experiment 3). Frozen embryos as nuclear donor cells worked equally well as fresh embryos for cloning in embryo development which was superior to IVF embryos (Experiment 4). However, fresh embryos resulted in a higher proportion (P < 0.05) of blastomere recovery than did frozen of IVF embryos. Finally, embryo transfer of cloned embryos from our procedure produced a viable calf, demonstrating the commercial value of this novel approach of the technology.
Mol Reprod Dev 1993 May
PMID:Nuclear transfer in cattle: effect of nuclear donor cells, cytoplast age, co-culture, and embryo transfer. 850 77


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