To investigate whether sperm entry point
(SEP) is random or preferential in bovine,
oocytes (n=311) were submitted to in vitrofertilization (IVF) as described previously (10 (link)).
In brief, frozen-thawed and washed sperm from
a single Holstein sire of proven in vitro fertility
were used for fertilization after capacitation by
the swim-up procedure. Spermatozoa (1×106/
ml sperm) and matured COCs (40-45 COCs/200
μl) were co-incubated in modified fertilization-
Tyrode’s albumin lactate pyruvate (TALP,
handmade) medium containing 0.01 mM
heparin (Sigma, USA), 0.2 mM penicillamine
(Sigma, USA) and 0.1 mM hypotaurine
(Sigma, USA) for 18 hours at 39.5˚C, 6%
CO2 in humidified air. Fertilized oocytes at
4-5 hours post fertilization (hpf) were fixed,
stained with Hoechst-33342 and visualized
with a fluorescence-assisted micromanipulator
microscope (Olympus, Japan). Schurmann et al.
(14 (link)) demonstrated 4-6 hpf as the optimum timepoint
of maximum early fertilization. To map
the SEP, eggs were rotated under constant UVlight
until MII-spindle (the approved reference
point of SEP) was positioned to 3 O’clock.
Then, the spatial relationship between SEP and MII-spindle was measured as described
by Motosugi et al. (Fig .2 ) (15 (link)). In this scheme,
zones I and IV are the closest and the furthest
from the MII-spindle, respectively.
(SEP) is random or preferential in bovine,
oocytes (n=311) were submitted to in vitrofertilization (IVF) as described previously (10 (link)).
In brief, frozen-thawed and washed sperm from
a single Holstein sire of proven in vitro fertility
were used for fertilization after capacitation by
the swim-up procedure. Spermatozoa (1×106/
ml sperm) and matured COCs (40-45 COCs/200
μl) were co-incubated in modified fertilization-
Tyrode’s albumin lactate pyruvate (TALP,
handmade) medium containing 0.01 mM
heparin (Sigma, USA), 0.2 mM penicillamine
(Sigma, USA) and 0.1 mM hypotaurine
(Sigma, USA) for 18 hours at 39.5˚C, 6%
CO2 in humidified air. Fertilized oocytes at
4-5 hours post fertilization (hpf) were fixed,
stained with Hoechst-33342 and visualized
with a fluorescence-assisted micromanipulator
microscope (Olympus, Japan). Schurmann et al.
(14 (link)) demonstrated 4-6 hpf as the optimum timepoint
of maximum early fertilization. To map
the SEP, eggs were rotated under constant UVlight
until MII-spindle (the approved reference
point of SEP) was positioned to 3 O’clock.
Then, the spatial relationship between SEP and MII-spindle was measured as described
by Motosugi et al. (
zones I and IV are the closest and the furthest
from the MII-spindle, respectively.
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