Julieta L Rojo1, Martina Linari1, Kelly A Young2, Marina C Peluffo3. 1. Centro de Investigaciones Endocrinológicas "Dr. César Bergadá" (CEDIE), CONICET, FEI, División de Endocrinología Hospital de Niños Ricardo Gutiérrez, Gallo 1330, CABA, C1425EFD, Buenos Aires, Argentina. 2. Department of Biological Sciences, California State University Long Beach, Long Beach, CA, 90840, USA. 3. Centro de Investigaciones Endocrinológicas "Dr. César Bergadá" (CEDIE), CONICET, FEI, División de Endocrinología Hospital de Niños Ricardo Gutiérrez, Gallo 1330, CABA, C1425EFD, Buenos Aires, Argentina. mpeluffo@cedie.org.ar.
Abstract
PURPOSE: We hypothesized that the chemokine SDF1/CXCR4 system was present in feline cumulus-oocyte complexes (COCs) and that COCs cultured with SDF1 would directly upregulate gene expression in the ovulatory cascade. METHODS: Ovaries (n = 50) were obtained from adult domestic cats during the breeding season and COCs were recovered from antral follicles. Because IVM media triggers cumulus-oocyte expansion, culture conditions needed to be optimized to study periovulatory genes. After optimization, the effects of 25 ng/ml SDF1 and the CXCR4 inhibitor were examined in a COC culture for 3, 12, and 24 h. RESULTS: MEM-hepes with 1% of charcoal stripped-FBS was the optimized culture medium, assessed by the expansion of COCs at 24 h in the gonadotropin (GNT) group but not in the media with serum alone. The mRNA expression of HAS2, TNFAIP6, PTX3, and AREG peaked at 3 h in GNT group as compared to all other groups (p < 0.05). COCs cultured with SDF1 showed increased HAS2 and TNFAIP6 mRNA expression at 3 h compared to negative controls and to the CXCR4 inhibitor group. CXCR4 and SDF1 immunostaining was present in both cumulus cells and the oocyte. CONCLUSIONS: These results demonstrate that GNT stimulation upregulates key periovulatory genes and expansion in feline COCs from antral follicles, and support the use of this culture system to examine molecular processes within the COC. In addition, SDF1 directly promotes key periovulatory genes in feline COCs, suggesting that the SDF1-CXCR4 pathway may extend its function beyond a chemoattractant, and may play a direct role within the COC.
PURPOSE: We hypothesized that the chemokine SDF1/CXCR4 system was present in feline cumulus-oocyte complexes (COCs) and that COCs cultured with SDF1 would directly upregulate gene expression in the ovulatory cascade. METHODS: Ovaries (n = 50) were obtained from adult domestic cats during the breeding season and COCs were recovered from antral follicles. Because IVM media triggers cumulus-oocyte expansion, culture conditions needed to be optimized to study periovulatory genes. After optimization, the effects of 25 ng/ml SDF1 and the CXCR4 inhibitor were examined in a COC culture for 3, 12, and 24 h. RESULTS: MEM-hepes with 1% of charcoal stripped-FBS was the optimized culture medium, assessed by the expansion of COCs at 24 h in the gonadotropin (GNT) group but not in the media with serum alone. The mRNA expression of HAS2, TNFAIP6, PTX3, and AREG peaked at 3 h in GNT group as compared to all other groups (p < 0.05). COCs cultured with SDF1 showed increased HAS2 and TNFAIP6 mRNA expression at 3 h compared to negative controls and to the CXCR4 inhibitor group. CXCR4 and SDF1 immunostaining was present in both cumulus cells and the oocyte. CONCLUSIONS: These results demonstrate that GNT stimulation upregulates key periovulatory genes and expansion in feline COCs from antral follicles, and support the use of this culture system to examine molecular processes within the COC. In addition, SDF1 directly promotes key periovulatory genes in feline COCs, suggesting that the SDF1-CXCR4 pathway may extend its function beyond a chemoattractant, and may play a direct role within the COC.
Authors: M C Peluffo; J Stanley; N Braeuer; A Rotgeri; K-H Fritzemeier; U Fuhrmann; B Buchmann; T Adevai; M J Murphy; M B Zelinski; B Lindenthal; J D Hennebold; R L Stouffer Journal: Hum Reprod Date: 2014-04-29 Impact factor: 6.918
Authors: L J McKenzie; S A Pangas; S A Carson; E Kovanci; P Cisneros; J E Buster; P Amato; M M Matzuk Journal: Hum Reprod Date: 2004-10-07 Impact factor: 6.918