This study was performed to investigate the effect of melatonin on bovine oocyte maturation and subsequent embryonic development in vitro. The endogenous melatonin concentration in bovine follicular fluid is approximately 10(-11) M. To examine the potential beneficial effects of melatonin on bovine oocyte maturation in vitro, germinal vesicle (GV) oocytes were incubated with different concentrations of melatonin (10(-11), 10(-9), 10(-7), 10(-5), 10(-3) M). Melatonin supplementation at suitable concentrations significantly promoted oocyte maturation. The development of embryos and the mean cell number/blastocyst produced after in vitro fertilization were remarkably improved. The most effective melatonin concentrations obtained from the studies ranged from 10(-9) to 10(-7) M. The expression of melatonin receptor MT1 and MT2 genes was identified in cumulus cells, granulosa cells, and oocytes using reverse transcription PCR, immunofluorescence, and Western blot. The mechanistic studies show that the beneficial effects of melatonin on bovine oocyte maturation are mediated via melatonin membrane receptors as the melatonin receptor agonist (IIK7) promotes this effect while the melatonin receptor antagonist (luzindole) blocks this action. Mechanistic explorations revealed that melatonin supplementation during bovine oocyte maturation significantly up-regulated the expressions of oocyte maturation-associated genes (GDF9, MARF1, and DNMT1a) and cumulus cells expansion-related gene (PTX3, HAS1/2) and that LHR1/2, EGFR are involved in signal transduction and epigenetic reprogramming. The results obtained from the studies provide new information regarding the mechanisms by which melatonin promotes bovine oocyte maturation in vitro and provide an important reference for in vitro embryo production of bovine and the human-assisted reproductive technology.
Profilin is an actin-binding protein that shows complex effects on the dynamics of the actin cytoskeleton. There are five profilin isoforms in Arabidopsis thaliana L. However, it is still an open question whether these isoforms are functionally different. In the present study, two profilin isoforms from Arabidopsis, PRF1 and PRF2 were fused with green fluorescent protein (GFP) tag and expressed in Escherichia coli and A. thaliana in order to compare their biochemical properties in vitro and their cellular distributions in vivo. Biochemical analysis revealed that fusion proteins of GFP-PRF1 and GFP-PRF2 can bind to poly-L-proline and G-actin showing remarkable differences. GFP-PRF1 has much higher affinities for both poly-L-proline and G-actin compared with GFP-PRF2. Observations of living cells in stable transgenic A. thaliana lines revealed that 35S::GFP-PRF1 formed a filamentous network, while 35S::GFP-PRF2 formed polygonal meshes. Results from the treatment with latrunculin A and a subsequent recovery experiment indicated that filamentous alignment of GFP-PRF1 was likely associated with actin filaments. However, GFP-PRF2 localized to polygonal meshes resembling the endoplasmic reticulum. Our results provide evidence that Arabidopsis profilin isoforms PRF1 and PRF2 have different biochemical affinities for poly-L-proline and G-actin, and show distinctive localizations in living cells. These data suggest that PRF1 and PRF2 are functionally different isoforms.
Studies of the living embryo sacs of Torenia fournieri reveal that the actin cytoskeleton undergoes dramatic changes that correlate with nuclear migration within the central cell and the primary endosperm. Before pollination, actin filaments appear as short bundles randomly distributed in the cortex of the central cell. Two days after anthesis, they become organized into a distinct actin network. At this stage the secondary nucleus, which is located in the central region of the central cell, possesses an associated array of short actin filaments. Soon after pollination, the actin filaments become fragmented in the micropylar end and the secondary nucleus is located next to the egg apparatus. After fertilization, the primary endosperm nucleus moves away from the egg cell and actin filaments reorganize into a prominent network in the cytoplasm of the primary endosperm. Disruption of the actin cytoskeleton with latrunculin A and cytochalasin B indicates that actin is involved in the migration of the nucleus in the central cell. Our data also suggest that the dynamics of actin cytoskeleton may be responsible for the reorganization of the central cell and primary endosperm cytoplasm during fertilization.
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