Deficiencies in extrusion of the second polar body due to high calcium concentrations during in vitro fertilization in inbred C3H/He mice.
Successful in vitro fertilization (IVF) of all inbred strains of laboratory mice has not yet been accomplished. We have previously shown that a high calcium concentration improved IVF in various inbred mice. However, we also found that in cumulus-free ova of C3H/He mice such IVF conditions significantly increased the deficiency of extrusion of the second polar body (PBII) in a dose-dependent manner (2% at 1.71 mM and 29% at 6.84 mM, P < 0.05) and that PBII extrusion was affected by high calcium levels at 2-3 h post-insemination. While developmental competence of ova without PBII extrusion to blastocysts after 96 h culture was not affected, a significant reduction in the nuclear number of the inner cell mass was observed in blastocyst fertilized under high calcium condition. We also examined how high calcium concentration during IVF affects PBII extrusion in C3H/He mice. Cumulus cells cultured under high calcium conditions showed a significantly alleviated deficient PBII extrusion. This phenomenon is likely to be specific to C3H/He ova because deficient PBII extrusion in reciprocal fertilization between C3H and BDF1 gametes was observed only in C3H/He ova. Sperm factor(s) was still involved in deficient PBII extrusion due to high calcium concentrations, as this phenomenon was not observed in ova activated by ethanol. The cytoskeletal organization of ova without PBII extrusion showed disturbed spindle rotation, incomplete formation of contractile ring and disturbed localization of actin, suggesting that high calcium levels affect the anchoring machinery of the meiotic spindle. These results indicate that in C3H/He mice high calcium levels induce abnormal fertilization, i.e. deficient PBII extrusion by affecting the cytoskeletal organization, resulting in disturbed cytokinesis during the second meiotic division. Thus, use of high calcium media for IVF should be avoided for this strain.
Science Service Inc.,4-9-1 Anagawa Inage-ku,Chiba,263-8555,Japan.
Yuki
Y
Ohta
University Farm,Faculty of Agriculture,Utsunomiya University,Shimokomoriya 443,Mohka,Tochigi 321-4415,Japan.
Yoshikazu
Y
Nagao
Graduate School of Agriculture,Kyoto University,Kitashirakawa Oiwake-cho,Sakyo-ku,Kyoto 606-8502,Japan.
Naojiro
N
Minami
Department of Technical Support and Development,National Institute of Radiological Sciences,4-9-1 Anagawa Inage-ku,Chiba,263-8555,Japan.
Satoshi
S
Tsukamoto
Department of Technical Support and Development,National Institute of Radiological Sciences,4-9-1 Anagawa Inage-ku,Chiba,263-8555,Japan.
Seiji
S
Kito
D000818:Animals; D001755:Blastocyst / Q000378:metabolism; D053624:Blastocyst Inner Cell Mass / Q000166:cytology / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D005260:Female; D005306:Fertilization; D005307:Fertilization in Vitro / Q000379:methods*; D008297:Male; D008809:Mice, Inbred C3H; D018613:Microscopy, Confocal; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D059705:Polar Bodies / Q000378:metabolism*; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism
D002118:Calcium
C3H/He mice; Calcium and cytoskeletal organization; In vitro fertilization; Inbred; Ova; Second polar body