Expression and regulation of stromal cell-derived factor-1 (SDF1) and chemokine CXC motif receptor 4 (CXCR4) in equine and bovine preovulatory follicles.
The interaction between stromal cell-derived factor-1 (SDF1) and chemokine CXC motif receptor 4 (CXCR4) has been implicated in leukocyte attraction, tissue remodeling and angiogenesis. The objective of the present study was to characterize the expression and regulation of SDF1 and CXCR4 in equine follicles during the ovulatory process. Equine preovulatory follicles were isolated during estrus 0-39h after hCG treatment. Follicle wall preparations (theca interna with attached granulosa cells) and isolated preparations of granulosa cells and theca interna were obtained, and total RNA extracts were analyzed by RT-PCR/Southern blot. Results showed that levels of CXCR4 transcripts were induced by hCG in follicles at 36 h post-hCG (P<0.05 vs 0 h), with the induction observed in both granulosa and theca cells. Immunoblotting and immunohistochemical analyses confirmed an increase in CXCR4 protein in follicles after hCG treatment. In contrast, levels of SDF1 transcripts were very low in granulosa cells but high in theca interna cells throughout most of the ovulatory period. Studies in vivo performed with bovine preovulatory follicles collected 0-24h post-hCG revealed a marked and significant up-regulation of CXCR4 transcripts after hCG (P<0.05), as observed in equine follicles. A similar pattern of CXCR4 mRNA up-regulation was observed in cultures of bovine granulosa cells treated with forskolin (P<0.05). This forskolin-dependent induction of CXCR4 mRNA was suppressed by co-treatment with inhibitors of PKA, ERK1/2 and EGFR, and by the progesterone receptor antagonist RU486 (P<0.05), underscoring the contribution of multiple signaling pathways. In complementary studies, treatment of bovine granulosa cells with EGF or the hypoxia mimetic cobalt chloride significantly increased CXCR4 transcript levels, whereas co-treatment with forskolin and a CXCR4 antagonist repressed the expression of several ovulation-related genes. Collectively, this study describes for the first time the gonadotropin-dependent up-regulation of CXCR4 transcript in ovarian follicles of large monoovulatory species, provides some insights into the regulation of CXCR4 gene expression in granulosa cells, and identifies a potential link between follicular SDF1/CXCR4 activation and the regulation of ovulation-related genes.
Centre de Recherche en Reproduction Animale, Université de Montréal, Saint-Hyacinthe, Québec J2S 7C6, Canada; Département de Biomédecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec J2S 7C6, Canada; Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec J2S 7C6, Canada. Electronic address: k.sayasith@umontreal.ca.
Khampoun
K
Sayasith
Centre de Recherche en Reproduction Animale, Université de Montréal, Saint-Hyacinthe, Québec J2S 7C6, Canada; Département de Biomédecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec J2S 7C6, Canada; Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec J2S 7C6, Canada.
Jean
J
Sirois
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D054377:Chemokine CXCL12 / Q000235:genetics* / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000008:administration & dosage; D003035:Cobalt / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004971:Estrus / Q000502:physiology; D005260:Female; D005786:Gene Expression Regulation; D006107:Granulosa Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006736:Horses; D006801:Humans; D015735:Mifepristone / Q000494:pharmacology; D008969:Molecular Sequence Data; D010060:Ovulation / Q000502:physiology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D019718:Receptors, CXCR4 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D013799:Theca Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D054377:Chemokine CXCL12; D006063:Chorionic Gonadotropin; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D019718:Receptors, CXCR4; D005576:Colforsin; D015735:Mifepristone; D003035:Cobalt; D004815:Epidermal Growth Factor; C018021:cobaltous chloride
And ovulation-related genes; Chemokine receptor; Gonadotropin; Ovulatory process; Preovulatory follicles; Primary granulosa cells