Effects of follicular cells and FSH on the resumption of meiosis in equine oocytes matured in vitro.
It has been suggested that preculturing immature oocytes in a manner that maintains them in meiotic arrest may improve cytoplasmic maturation and, thereby, the eventual developmental competence of oocytes matured in vitro. This study examined the ability of follicular cells to maintain meiotic arrest in equine oocytes. Cumulus-oocyte complexes (COCs) recovered from dead mares were cultured for 38 h in M199 either attached to, or together with, different follicle wall components, as follows: (1) attached to the follicle wall, (2) cocultured with separated follicle wall, (3) attached to membrana granulosa (COCG), (4) COCGs cocultured with sheets of theca cells, (5) COCGs cultured in theca-cell conditioned medium, and (6) control COCs without any follicle wall components. When oocytes were cultured attached to their follicle wall, 79% remained in the GV stage throughout the 38 h incubation. However, when oocytes were cocultured with separate pieces of follicle wall, meiosis resumed and a similar proportion of oocytes progressed to metaphase II (79%) as under control conditions (84%). Only 16% of oocytes cultured while still attached to the membrana granulosa (COCGs) maintained the GV stage, whereas when COCGs were cocultured with theca cells or in theca-cell conditioned medium, significantly more oocytes remained in the GV stage (64 and 52%, respectively), indicating that theca cells secrete a meiosis-inhibiting factor. The effect of FSH on the meiosis-inhibiting activity of follicular cells was investigated by culturing COCs attached to the follicle wall and COCGs in the presence or absence of theca cells in medium containing FSH. Addition of 0.05 iu recombinant human FSH ml(-1) to the culture medium did not affect nuclear maturation and failed to overcome the suppressive effect exerted by the follicle wall or by theca cells, despite the fact that mRNA for the FSH receptor was found using RT-PCR in both cumulus and granulosa cells. These results demonstrate that the maintenance of meiotic arrest in equine oocytes during culture can be promoted by theca cells, which appear to act via a secreted inhibitory factor that cannot be suppressed or counteracted by FSH.
Tremoleda
J L
JL
Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, The Netherlands. J.Tremoleda@vet.uu.nl
Tharasanit
T
T
Van Tol
H T A
HT
Stout
T A E
TA
Colenbrander
B
B
Bevers
M M
MM
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D018920:Coculture Techniques; D017077:Culture Media, Conditioned; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D006107:Granulosa Cells / Q000502:physiology; D006736:Horses / Q000502:physiology*; D008540:Meiosis / Q000187:drug effects*; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000166:cytology*; D006080:Ovarian Follicle / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011962:Receptors, FSH / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013799:Theca Cells / Q000502:physiology
D017077:Culture Media, Conditioned; D012333:RNA, Messenger; D011962:Receptors, FSH; D005640:Follicle Stimulating Hormone