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Stromal-derived factor 1 directly promotes genes expressed within the ovulatory cascade in feline cumulus oocyte complexes

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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.

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Acknowledgements

We are grateful to Olga Bustamante from the “Centro de Sanidad Animal de la Municipalidad de Merlo” (Provincia de Buenos Aires) for the donation of the feline ovaries. Recombinant human FSH and LH (Merck Serono) were generously donated for this project. A special thanks to German La Iacona for his technical assistance with the confocal microscope.

Funding

This study was supported PRESTAMO BID PICT 2014 N° 666 (MCP), Small Faculty Grants Program CNSM CSULB (KAY), the CSULB Professors Around the World Award (KAY), and by the Fogarty International Center, of the National Institutes of Health under Award R01TW009163 (MCP).

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Authors and Affiliations

  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

    Julieta L. Rojo, Martina Linari & Marina C. Peluffo

  2. Department of Biological Sciences, California State University Long Beach, Long Beach, CA, 90840, USA

    Kelly A. Young

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  1. Julieta L. Rojo
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  2. Martina Linari
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Correspondence to Marina C. Peluffo.

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Electronic supplementary material

Online Resource 1

COCs cultured for 12 h with 25 ng/ml SDF1 showed no changes (P > 0.05) in (a) HAS2, (b) TNFAIP6, (c) PTX3, (d) AREG and (e) GDF9 expression at 12 h between SDF1 and control treated groups. Bars represent the real-time PCR relative-quantitation of mRNA levels normalized to 18S (mean ± SEM; n = 4/time point). P values for the analysis of each gene are listed in each corresponding graph. (PDF 38 kb)

Online Resource 2

COCs cultured for 24 h with 25 ng/ml SDF1 showed no changes (P > 0.05) in (a) HAS2, (b) TNFAIP6, (c) PTX3, (d) AREG and (e) GDF9 expression at 24 h between SDF1 and control treated groups. Bars represent the real-time PCR relative-quantitation of mRNA levels normalized to 18S (mean ± SEM; n = 4/time point). P values for the analysis of each gene are listed in each corresponding graph. (PDF 40 kb)

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Rojo, J.L., Linari, M., Young, K.A. et al. Stromal-derived factor 1 directly promotes genes expressed within the ovulatory cascade in feline cumulus oocyte complexes. J Assist Reprod Genet 35, 785–792 (2018). https://doi.org/10.1007/s10815-018-1150-4

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  • DOI: https://doi.org/10.1007/s10815-018-1150-4

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