Effect of medium milieu on sperm penetration and pronuclear formation of bovine oocytes matured in vitro. Byung-Ki Kima, Sang-Chan Leeb, Kwang-Sun Leea, Bok-Kyu Leea, Chang-Hee Hana, Jong-Heung Kimb, Chae-Sik Leeb
Theriogenology 57:2093-2104, 2002
Effect of medium milieu on sperm penetration
and pronuclear formation of bovine
oocytes matured in vitro
Byung-Ki Kima, Sang-Chan Leeb, Kwang-Sun Leea,
Bok-Kyu Leea, Chang-Hee Hana, Jong-Heung Kimb,
Chae-Sik Leeb
aFaculty of Natural Science, Department of Biology,
University of Dong-Eui,Kaya-Dong, Pusan 614-714,
South Korea, bSaewha Women's Clinic, Pusan, South Korea
Abstract
The purpose of the present study was to investigate the optimal
concentration of osmolarity, calcium and bicarbonate for sperm penetration
and formation of pronuclei(PN), and to investigate the time required for
capacitation, penetration across the zona pellucida and formation of PN in
bovine cumulus-free oocytes matured in vitro. Bovine follicular oocytes
collected at slaughter were matured and fertilized in vitro. Bovine sperm
penetrated the zona pellucida in medium containing 240 to 440 mOsm,
whereas PN formation was observed in a narrow range of osmolarities, from
280 to 360 mOsm. Maximal penetration by spermatozoa and PN formation was
obtained in the medium with 2.5 mM calcium. High rates of spermatozoa
penetration were observed in the medium with 37 to 49 mM NaHCO3. However,
PN were formed regardless of the concentration of NaHCO3. The times
required for sperm capacitation and penetration through the zona pellucida
were 260 and 50 min, respectively. The first development of PN was
recorded at 120 min after sperm penetration. Therefore, our study suggests
that fertilization ability of spermatozoa in vitro appears to be more
stable in high concentrations of NaCl. Oocytes are more sensitive to
osmotic stress than spermatozoa. Calcium is required for both sperm
penetration and PN formation in cumulus-free oocytes, but bicarbonate may
be needed mainly for the penetration of spermatozoa.
Key words: Osmolarity; Calcium; Bicarbonate; Pronuclei