Molecular characterization of tumor necrosis alpha-induced protein 6 and its human chorionic gonadotropin-dependent induction in theca and mural granulosa cells of equine preovulatory follicles.
The preovulatory rise in gonadotropins causes an expansion of the cumulus-oocyte complex, a process requiring the induction of several genes. The objectives of this study were to clone the equine tumor necrosis factor alpha-induced protein 6 (TNFAIP6), and investigate its regulation in equine follicles during human chorionic gonadotropin (hCG)-induced ovulation. The isolation of the equine TNFAIP6 cDNA revealed that it contains an open reading frame of 834 bp (including the stop codon), encoding a predicted 277 amino acid protein that is highly similar (91-93% identity) to known mammalian homologs. The regulation of TNFAIP6 mRNA was studied in equine follicles isolated during estrus between 0 and 39 h post-hCG and in corpora lutea (CL) obtained on day 8 of the estrous cycle. Results from semi-quantitative RT-PCR/Southern blot showed that levels of TNFAIP6 mRNA were low in follicles obtained at 0 h, increased at 12 h, returned to basal levels at 24 h, and increased again at 36 h post-hCG (P<0.05). Levels of TNFAIP6 transcripts were relatively moderate in CL, but low in non-ovarian tissues tested. Analyses performed with isolated preparations of theca and granulosa cells indicated that TNFAIP6 mRNA was regulated in both layers, with a maximal induction obtained 33-36 h post-hCG (P<0.05). Immunohistochemical staining of sections of equine follicles isolated at 0 and 33 h post-hCG confirmed the induction of TNFAIP6 protein in both cell types after hCG treatment. Thus, the present study describes for the first time the gonadotropin-dependent regulation of follicular TNFAIP6 during the ovulation in a monoovulatory species. The biphasic induction of TNFAIP6 in equine theca and granulosa cells differs from the pattern observed in rodents, suggesting a distinct control of gene expression in this monoovulatory species.
Sayasith
Khampoune
K
Centre de Recherche en Reproduction Animale, Université de Montréal, Canada. k.sayasith@umontreal.ca
Doré
Monique
M
Sirois
Jean
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002417:Cattle; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D003338:Corpus Luteum / Q000737:chemistry; D005260:Female; D005498:Follicular Phase; D006107:Granulosa Cells / Q000737:chemistry*; D006736:Horses / Q000378:metabolism*; D006801:Humans; D018960:Hyaluronan Receptors / Q000032:analysis / Q000235:genetics*; D007150:Immunohistochemistry; D051379:Mice; D008969:Molecular Sequence Data; D010062:Ovulation Induction; D012333:RNA, Messenger; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017385:Sequence Homology; D013799:Theca Cells / Q000737:chemistry*
D006063:Chorionic Gonadotropin; D018960:Hyaluronan Receptors; D012333:RNA, Messenger