D. Huneau et al., ESTROUS SHEEP SERUM AS A POTENT AGENT FOR OVINE IVF - EFFECT ON CHOLESTEROL EFFLUX FROM SPERMATOZOA AND THE ACROSOME REACTION, Theriogenology, 42(6), 1994, pp. 1017-1028
The aim of the present study was to evaluate the effect of heat-inacti
vated estrous sheep serum (ESS) on sheep IVF. When the capacitation an
d the fertilization media contained 20% ESS, a fertilization rate of 8
% was achieved. The beneficial effect of ESS on sheep IVF was further
demonstrated since the fertilization rate was null when ESS was omitte
d during sperm capacitation and fertilization. Estrous sheep serum sup
ported both sperm capacitation and fertilization as shown by the resul
ts of experiments in which it was omitted during one of these steps: s
perm capacitation in serum-free medium resulted in delayed sperm-oocyt
e penetration, while fertilization in serum-free medium significantly
decreased the percentage of fertilized oocytes. To investigate the inf
luence of serum on sperm ability to undergo the acrosome reaction, sal
t-stored follicular sheep oocytes were inseminated, and the acrosomal
status of spermatozoa attached to zonae was examined by electron micro
scopy after a 4-h period of coincubation. Quantitative analysis on thi
n sections demonstrated that fewer acrosome-reacted spermatozoa were o
bserved when the capacitation and insemination steps were carried out
in DM-H medium without serum than in DM-H-SS supplemented with 20% ESS
(0.08, [0; 0.34], (median, range)/100 mu m zona vs 1.32, [0.90; 2.28]
/100 mu m zona; P < 0.01). Since a higher number of spermatozoa attach
ed to the zona surface in DM-H medium, the proportion of acrosome-reac
ted spermatozoa was much lower (0.7%, [0%; 2.2%], (median, range) vs 5
4%, [25%; 100%]; P < 0.01) in the absence of serum. These results indi
cate that in our IVF system the development of the acrosome reaction d
epended on serum. Sperm cholesterol efflux during in vitro capacitatio
n was measured on [H-3]cholesterol labeled spermatozoa resuspended in
DM-H or DM-H-SS medium. A time-dependent cholesterol removal was obser
ved in the presence of serum (60 +/- 5%, mean +/- SD, after 5 h), wher
eas it was limited to 14 +/- 3 % in DM-H medium; hence addition of ser
um to the capacitation medium efficiently supports cholesterol efflux,
which is thought to be a key-event in the capacitation process.