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Ayumi HASEGAWA Keiji MOCHIDA Toshiko TOMISHIMA Kimiko INOUE Atsuo OGURA 《The Journal of reproduction and development》2014,60(3):187-193
Successful in vitro fertilization (IVF) in mice has been achieved using spermatozoa at concentrations
specifically optimized for the experimental conditions, such as species and source of spermatozoa. Although IVF in mice is
mostly performed using about 80–500 µl drops, it is expected that the number of spermatozoa used for insemination can be
reduced by decreasing the size of the IVF drops. The present study was undertaken to examine the extent to which the number
of spermatozoa used for IVF could be reduced by using small droplets (1 µl). We devised the experimental parameters using
frozen–thawed spermatozoa from C57BL/6 mice in anticipation of broader applications to other mouse facilities. We found that
as few as 5 spermatozoa per droplet could fertilize oocytes (1 or 3 oocytes per droplet), although the fertilization rates
were low (13–15%). Practical fertilization rates (> 40%) could be achieved with frozen-thawed C57BL/6J spermatozoa, which
are sensitive to cryopreservation, when 20 sperm per droplet were used to inseminate 3 oocytes. Even with spermatozoa from a
very poor quality suspension (10% motility), about 25% of oocytes were fertilized. Our calculations indicate that the number
of inseminated spermatozoa per oocyte can be reduced to 1/96–1/240 by this method. In two separate embryo transfer
experiments, 60% and 47%, respectively, of embryos developed to term. Our microdroplet IVF method may be particularly
advantageous when only a limited number of motile spermatozoa are available because of inadequate freezing-thawing or genetic
reasons. 相似文献
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During spermatogenesis, many proteins are synthesized in the testis prior to the completion of sperm maturation. Many components are involved in the testicular protein synthesis and one of the components that is implicated in the polypeptide chain elongation is elongation factor 1α. In the present study, the molecular cloning of elongation factor 1α (EF-1α) was conducted from a testis cDNA library of the Nile tilapia. The cDNA for tilapia EF-1α (tEF-1α) contains a complete open reading frame encoding 462 amino acids. The predicted amino acid sequence of EF-1α shows an approximate 90% similarity to those identified in other teleost fish, such as medaka, sea bream and zebrafish. Northern blotting revealed that the gene is expressed in all of the examined tissues and in ovulated eggs. The results of in situ hybridization indicate that the gene is expressed specifically in Leydig cells in testis, suggesting the involvement of EF-1α as an actin-binding protein in the cluster formation of Leydig cells. In the ovary, the gene is expressed in the perinucleolus stage of oocytes, suggesting that EF-1α is also implicated in oocyte growth. 相似文献
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