N. L. First

Bio: N. L. First is an academic researcher from University of Wisconsin-Madison. The author has contributed to research in topics: Sperm & Human fertilization. The author has an hindex of 50, co-authored 157 publications receiving 11034 citations. Previous affiliations of N. L. First include Institut national de la recherche agronomique.

Capacitation of bovine sperm by heparin.

Abstract: Capacitation of bovine sperm was evaluated by determining the ability of sperm to fertilize bovine oocytes in vitro and to undergo an acrosome reaction upon exposure to lysophosphatidylcholine (LC). Incubation of sperm with heparin (10 micrograms/ml) increased the percentage of oocytes fertilized, but this required exposing sperm to heparin for at least 4 h before adding them to oocytes. There was no effect on the percentage of motile or acrosome-reacted sperm after exposure of noncapacitated sperm to 100 micrograms/ml LC for 15 min. When sperm were incubated for 4 h with heparin, exposure to 100 micrograms/ml LC for 15 min had no effect on the percentage of sperm that were motile, but the percentage of acrosome-reacted sperm increased from less than 10% to over 70%. The acrosome reactions (ARs) induced by LC were synchronous, reached maximal levels within 15 min, and differed (p less than 0.001) between sperm incubated under capacitating (with heparin) and noncapacitating conditions (without heparin). The time course required for heparin to capacitate sperm as judged by in vitro fertilization and to render sperm sensitive to LC induction of the AR were found to be similar. The percentage of ARs induced by LC and percentage of oocytes fertilized by sperm were found to be heparin-dose-dependent, with the maximum responses occurring at 5-10 micrograms/ml heparin. The correlation between the mean fertilization and LC-induced AR percentages was 0.997 (p less than 0.01). These studies demonstrate capacitation of bovine sperm by heparin requires at least a 4-h exposure of sperm to heparin and suggest that plasma membrane changes prior to an AR can be detected by exposure of bovine sperm to LC.

Development of bovine embryos in vitro as affected by energy substrates.

Abstract: Simple media were developed to study the metabolic requirements of bovine embryos up to Day 7 (Day 0 = day of oocyte aspiration) in vitro. Embryos were derived from oocytes matured and fertilized in vitro. At 45 +/- 2 h post insemination, embryos (> or = 2 cells) were randomly allotted to treatments. Examined in experiments 1 and 3 was the effect of pyruvate concentration in the presence of lactate. In the presence of lactate, pyruvate (0.2-5.0 mM) had no effect (p > 0.05) on the percentage of morulae or blastocysts. However, increasing the concentration of hemicalcium L-lactate from 5 mM to 10 mM decreased (p 0.05) on embryo development (experiment 4). However, the interaction of adding calcium and lactate resulted in a decreased (p < 0.05) percentage of morulae. Determined in experiment 6 were the independent effects of pyruvate, lactate, and glucose on embryo development in vitro. As pyruvate or lactate level was increased from 1 to 10 mM, the percentage of blastocysts was decreased (p < 0.05). These experiments indicate that adding pyruvate to a medium containing lactate is not necessary for development of bovine embryos in vitro.

Capacitation of bovine sperm by heparin: inhibitory effect of glucose and role of intracellular pH.

Abstract: Bovine sperm incubated with heparin for 7.5-8.5 h underwent an acrosome reaction in the absence but not the presence of glucose (5 mM). When sperm were incubated under capacitating conditions with heparin for 4 h, glucose inhibited sperm penetration of oocytes (p less than 0.01) and lysophosphatidylcholine (LC) induced acrosome reactions. Addition of glucose for the last 0.25 h of a 4.25-h incubation with heparin had no effect on ability of sperm to acrosome-react in response to LC. Nonmetabolizable sugars 3-O-methyl glucose, 2-deoxyglucose, sucrose, and sorbitol did not inhibit capacitation as judged by sperm sensitivity to LC or fertilization (p greater than 0.05), but capacitation was inhibited by the glycolyzable substrates glucose, mannose, and fructose (p less than 0.05). The glycolytic inhibitor, fluoride, reversed glucose inhibition of capacitation in a dose-dependent manner similar to its effect on glucose uptake by sperm. Extracellular pH declined from 7.4 to 7.2 during a 4-h incubation of sperm with heparin and glucose. The decline of extracellular pH during sperm incubation with glucose did not affect capacitation, since only an extracellular pH below 7.02 inhibited capacitation. The intracellular pH (pHi) of sperm increased 0.40 units over a 5-h incubation under capacitating conditions. The change in pHi was inhibited by glucose. Incubation of sperm with heparin and glucose for 12 h resulted in capacitated sperm as judged by both LC sensitivity and fertilizing ability. These studies demonstrate that glycolyzable substrates delay capacitation of bovine sperm and suggest the effect is in delaying an alkalinization of pHi.

Sheep cloned by nuclear transfer from a cultured cell line

Abstract: Nuclear transfer has been used in mammals as both a valuable tool in embryological studies and as a method for the multiplication of 'elite' embryos. Offspring have only been reported when early embryos, or embryo-derived cells during primary culture, were used as nuclear donors. Here we provide the first report, to our knowledge, of live mammalian offspring following nuclear transfer from an established cell line. Lambs were born after cells derived from sheep embryos, which had been cultured for 6 to 13 passages, were induced to quiesce by serum starvation before transfer of their nuclei into enucleated oocytes. Induction of quiescence in the donor cells may modify the donor chromatin structure to help nuclear reprogramming and allow development. This approach will provide the same powerful opportunities for analysis and modification of gene function in livestock species that are available in the mouse through the use of embryonic stem cells.

Capacitation of bovine sperm by heparin.

Abstract: Capacitation of bovine sperm was evaluated by determining the ability of sperm to fertilize bovine oocytes in vitro and to undergo an acrosome reaction upon exposure to lysophosphatidylcholine (LC). Incubation of sperm with heparin (10 micrograms/ml) increased the percentage of oocytes fertilized, but this required exposing sperm to heparin for at least 4 h before adding them to oocytes. There was no effect on the percentage of motile or acrosome-reacted sperm after exposure of noncapacitated sperm to 100 micrograms/ml LC for 15 min. When sperm were incubated for 4 h with heparin, exposure to 100 micrograms/ml LC for 15 min had no effect on the percentage of sperm that were motile, but the percentage of acrosome-reacted sperm increased from less than 10% to over 70%. The acrosome reactions (ARs) induced by LC were synchronous, reached maximal levels within 15 min, and differed (p less than 0.001) between sperm incubated under capacitating (with heparin) and noncapacitating conditions (without heparin). The time course required for heparin to capacitate sperm as judged by in vitro fertilization and to render sperm sensitive to LC induction of the AR were found to be similar. The percentage of ARs induced by LC and percentage of oocytes fertilized by sperm were found to be heparin-dose-dependent, with the maximum responses occurring at 5-10 micrograms/ml heparin. The correlation between the mean fertilization and LC-induced AR percentages was 0.997 (p less than 0.01). These studies demonstrate capacitation of bovine sperm by heparin requires at least a 4-h exposure of sperm to heparin and suggest that plasma membrane changes prior to an AR can be detected by exposure of bovine sperm to LC.

Primate embryonic stem cells

Abstract: A purified preparation of primate embryonic stem cells is disclosed. This preparation is characterized by the following cell surface markers: SSEA-1 (−); SSEA-4 (+); TRA-1-60 (+); TRA-1-81 (+); and alkaline phosphatase (+). In a particularly advantageous embodiment, the cells of the preparation are human embryonic stem cells, have normal karyotypes, and continue to proliferate in an undifferentiated state after continuous culture for eleven months. The embryonic stem cell lines also retain the ability, throughout the culture, to form trophoblast and to differentiate into all tissues derived from all three embryonic germ layers (endoderm, mesoderm and ectoderm). A method for isolating a primate embryonic stem cell line is also disclosed.