ABSTRACT
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.
Subject(s)
Animals, Genetically Modified/genetics , DNA/genetics , Gene Transfer Techniques , Spermatozoa , Animals , Animals, Genetically Modified/metabolism , Animals, Newborn , Blotting, Southern , Cattle , DNA/metabolism , DNA Restriction Enzymes/metabolism , Embryo, Mammalian/metabolism , Fertilization in Vitro , Green Fluorescent Proteins , In Vitro Techniques , Insemination, Artificial , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Male , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
Progesterone (P4) production by the bovine placenta differs from that of other steroidogenic tissue in two important respects: 1) it is calcium-dependent but cyclic nucleotide-independent and 2) it is suppressed by an endogenous inhibitor for most of the life span of the placenta. This natural refractory state of the placenta can be overcome in in vitro incubations of fetal cotyledon cells by agents that increase intracellular calcium (3-isobutylmethylxanthine [MIX], calcium ionophore (A23187), addition of substrate (pregnenolone, hydroxycholesterol), and stimulators of protein kinase C (PKC) such as phorbol ester (TPA). We therefore tested, in cultures of cotyledonary cells, two compounds that have been reported to inhibit protein kinases: 1) staurosporine (STA), an inhibitor of PKC, cAMP-dependent kinase, tyrosine kinase (TK), and the epidermal growth factor (EGF) receptor TK, and 2) genistein, an inhibitor of TK. It was found that STA stimulated steroidogenesis in a dose-dependent manner in both the absence and presence of added calcium. STA (10(-9) M) stimulated at least a twofold increase in P4 production by cultured fetal cotyledon cells throughout the first half of gestation (50-130 days). EGF was also found to cause a twofold stimulation of P4 production, and the effect was additive to that of STA. Both basal and EGF- or STA-stimulated production were inhibited by genistein. In contrast, two inhibitors of PKC and PKA (H-7, H-8) had no effect on P4 production. We conclude that STA-induced steroidogenesis in the bovine placenta is not related to its reported ability to inhibit PKC, TK, or EGF receptor TK.
Subject(s)
Alkaloids/pharmacology , Cattle/metabolism , Placenta/metabolism , Progesterone/biosynthesis , Animals , Calcium/pharmacology , Cells, Cultured , Cycloheximide/pharmacology , Female , Genistein , Isoflavones/pharmacology , Kinetics , Placenta/drug effects , Pregnancy , Protein Kinase Inhibitors , Protein Kinases/metabolism , StaurosporineABSTRACT
The oocyte-cumulus complex (25 oocytes per 250 microL medium) produced prostaglandin-F2 alpha (PGF2 alpha) and PGE2 during maturation, immediately following fertilization and for at least 48 h after fertilization. The data suggest that PG production is important in the development of the oocyte; addition of PGE2 (5 ng mL-1) to the fertilization medium increased the rate of cleavage and groups of oocytes with low cleavage rates produced far less PG than groups with high cleavage rates. Measurement of PG in the maturation medium could therefore be a means of assessing the suitability of oocytes for fertilization.