A multi-species meta-analysis of published transcriptomic data from models of oocyte competence identified the chromatin remodelling factor ATRX as a putative biomarker of oocyte competence. The objective of the current study was to test the hypothesis that ATRX protein expression by cumulus–oocyte complexes (COCs) reflects their intrinsic quality and developmental potential. In excess of 10,000 bovine COCs were utilised to test our hypothesis. COCs were in vitro matured (IVM) under conditions associated with reduced developmental potential: IVM in the presence or absence of (1) progesterone synthesis inhibitor (Trilostane); (2) nuclear progesterone receptor inhibitor (Aglepristone) or (3) an inducer of DNA damage (Staurosporine). ATRX protein expression and localisation were determined using immunocytochemistry and Western blot analysis. A proportion of COCs matured in the presence or absence of Trilostane was in vitro fertilised and cultured, and subsequent embryo development characteristics were analysed. In addition, ATRX expression was investigated in 40 human germinal vesicle-stage COCs. Our results showed that ATRX is expressed in human and bovine germinal vesicle oocytes and cumulus cells. In bovine, expression decreases after IVM. However, this decline is not observed in COCs matured under sub-optimal conditions. Blastocyst development rate and cell number are decreased, whereas the incidence of abnormal metaphase phase spindle and chromosome alignment are increased, after IVM in the presence of Trilostane (P < 0.05). In conclusion, localisation of ATRX to the cumulus cell nuclei and oocyte chromatin, after IVM, is associated with poor oocyte quality and low developmental potential. Furthermore, ATRX is dynamically regulated in response to progesterone signalling.
Lynne C O’Shea, Edward Daly, Carmel Hensey, and Trudee Fair
Noof Abdulrahman Alrabiah, Alexander C O Evans, Alan G Fahey, Niamh Cantwell, Patrick Lonergan, Janet McCormack, John A Browne, and Trudee Fair
Ovulation has been described as an inflammatory event, characterized by an influx of leukocytes into the ovulatory follicle and changes in the expression of immune factors in both the theca and granulosa tissue layers. Since information on this process is limited in cattle, our objective was to elucidate the contribution of the immune system to dominant follicle luteinization, ovulation and corpus luteum formation in cattle. Beef heifers (n=50) were oestrous synchronized, slaughtered and ovarian follicular or luteal tissue collected during a 96h window around ovulation. Follicular fluid cytokine concentration, temporal immune cell infiltration and inflammatory status were determined by Luminex multiplex analysis, immunohistochemistry and quantitative real time PCR-analysis, respectively, in pre- and peri-ovulatory follicular tissues. The concentrations of CXCL10 and VEGF-A were highest in pre-ovulatory follicular fluid samples. The pre and peri -ovulatory follicles play host to a broad repertoire of immune cells, including T-cells, granulocytes and monocytes. Dendritic cells were the most abundant cells in ovulatory follicular and luteal -tissue at all times. The mRNA expression of candidate genes associated with inflammation was highest in pre- and peri-ovulatory tissue, whereas tissue growth and modelling factors were highest in the post-ovulatory follicular and early luteal tissue. In conclusion, ovulation in cattle is characterized by the presence of neutrophils, macrophages and dendritic cells in the ovulatory follicle, reflected in compartmentalized cytokine and growth factor expression. These findings indicate a tightly regulated sterile inflammatory response to the LH surge in the ovulatory follicle which is rapidly resolved during early corpus luteum formation.
Sandra Bagés-Arnal, José M Sánchez, Beatriz Fernandez-Fuertes, Michael McDonald, Susanta K Behura, Thomas E Spencer, Trudee Fair, and Pat Lonergan
In cattle, embryo transfer into the uterine horn contralateral to the corpus luteum results in a higher incidence of pregnancy loss compared to transfer into the ipsilateral horn. We have previously reported temporal changes in the endometrial transcriptome during the estrous cycle which differ between uterine horns. The objective of this study was to compare the transcriptomic response of endometrium from the ipsilateral and contralateral horns to an elongating conceptus. Cross-bred beef heifers (n = 16) were synchronized and either used to generate day 14 conceptuses following the transfer of in vitro-produced blastocysts or to obtain day 14 endometrial explants. Conceptuses were recovered on day 14 by post-mortem uterine flushing, placed individually on top of explants collected from the ipsilateral (IPSI-D14) or the contralateral (CONTRA-D14) uterine horn of cyclic heifers, and co-cultured for 6 h. The response to a conceptus was markedly different between uterine horns, with 61 and 239 differentially expressed genes (DEGs; false discovery rate <0.05) in the ipsilateral and contralateral horns, respectively, compared to their controls. Direct comparison between IPSI-D1 and CONTRA-D14 revealed 32 DEGs, including CXCL11, CXCL10, IFIT2, RSAD2 and SAMD9. Gene Ontology analysis of these 32 genes revealed ten enriched biological processes, mainly related to immune response and response to an external stimulus. These data indicate that the endometrial response to the presence of a conceptus varies between uterine horns in the same uterus and may contribute to the higher incidence of pregnancy loss following embryo transfer to the contralateral horn.