Capacitation of Bovine Sperm by Heparin1

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.

Effect of supplementation of different growth factors in embryo culture medium with a small number of bovine embryos on in vitro embryo development and quality

“…For IVF, a final concentration of 1 3 10 6 sperm/ml was used. After in vitro maturation, COCs were washed three times in fertilisation Fert-Talp medium (Parrish et al, 1988) with 10 mg/ml of heparin (H9399) without epinephrine and hypotaurine and coincubated with the spermatozoa, in 500 ml using 4-Well Nunc R plates (Nunc, Fisher Scientific, Madrid, Spain), for 18 to 20 h in 5% CO 2 at 38.58C.…”

“…For IVF, a final concentration of 1 3 10 6 sperm/ml was used. After in vitro maturation, COCs were washed three times in fertilisation Fert-Talp medium (Parrish et al, 1988) with 10 mg/ml of heparin (H9399) without epinephrine and hypotaurine and coincubated with the spermatozoa, in 500 ml using 4-Well Nunc R plates (Nunc, Fisher Scientific, Madrid, Spain), for 18 to 20 h in 5% CO 2 at 38.58C.In vitro culture of embryos After IVF, presumptive zygotes were denuded from surrounding cumulus cells in HM199 and washed three times in CM, which was based on synthetic oviductal fluid aaci (Holm et al, 1999) with some modifications (Salvador et al, 2011) and with 3 mg/ml bovine serum albumin (A-8806) instead of bovine serum. Then, presumptive zygotes were in vitro cultured in groups of ,30 to 50 zygotes in 500 ml using 4-Well Nunc R plates, or in a number of five zygotes in 50 ml microdrops, depending on the experimental group (see below in Section 'Experimental design'), at 38.58C in a humidified atmosphere with 5% CO 2 and 5% O 2 in air.…”

Bicarbonate/CO2 induces rapid activation of phospholipase A2 and renders boar spermatozoa capable of undergoing acrosomal exocytosis in response to progesterone

“…Incubation in this medium results in no or very little loss of sperm viability (assessed by exclusion of propidium iodide [15]) or fertility [14] for up to 4 days. The Tyrode's medium used for incubation of spermatozoa was a modification of that used previously [11,16] and consisted of 102 mM NaCI, 3.1 mM KC1, 0.4 mM MgSO4, 0.3 mM NaHzPO4, 15 mM NaHCO3, 20 mM HEPES, 5 mM glucose, 16.7 mM sodium lactate, 1 mM sodium pyruvate, 20 gg phenol red/ml, 50 lag kanamycin/ml, and 3 mg bovine serum albumin (fraction V)/ml. The pyruvate and the albumin were added on the day of use.…”

Heterologous murine and bovine IVF using bottlenose dolphin (Tursiops truncatus) spermatozoa

“…(Nunc) (50 COCs in 250 µL of FERT medium) [26]. Frozen straws containing either dolphin or bovine spermatozoa were thawed in a water bath at 37 ºC for 50 s, and aliquots (5 µL) were taken for motility analysis by Computer assisted sperm Analysis,…”