Blastocysts exhibit sex-specific signalling of IFNT transcription, translation and activity

in Reproduction
Correspondence should be addressed to S E Ulbrich; Email: susanne.ulbrich@usys.ethz.ch

(M Büttner is now at Institute of Immunology, Veterinary College BBZ, University of Leipzig, Leipzig, Germany)

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Preimplantation bovine blastocyst supernatants exhibit sex-dependent antiviral activity, due to the ruminant pregnancy recognition signal interferon tau (IFNT). Differing potencies of IFNT variants have been supposed as cause, although evidence remains scarce. Here, we aimed at quantifying the sex-dependent IFNT production on transcriptional, translational and biological activity level in bovine blastocysts, to elucidate the origin of differences in antiviral activity between male and female blastocysts. Day 8 bovine blastocysts were co-cultured with endometrial stroma cells for 48 h. The embryonic IFNT mRNA expression was determined by quantitative reverse transcription followed by polymerase chain reaction (RT-qPCR). Additionally, the IFNT protein concentration was determined using a sensitive in-house developed IFNT-specific enzyme-linked immunosorbent assay (ELISA). The biological activity was assessed by quantifying the response of interferon-stimulated gene (ISG) expression in endometrial stroma cells. While the IFNT-specific ELISA displayed a limit of detection of 7.3 pg/mL, the stroma cell culture system showed to react to as little as 0.1 pg/mL IFNT in RT-qPCR analysis. The female blastocysts had a significant, 5.6-fold, 3.6-fold and 5.2-fold higher IFNT production than male blastocysts as determined by transcript abundance, protein concentration and, protein activity, respectively. Additionally, all parameters correlated positively, and therefore, we conclude that female blastocysts most likely have an increased IFNT gene and protein expression rather than expressing more potent IFNT variants.

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  • Supplement Table 1: IFNT-ELISA validation data for quality and reproducibility

 

    Society for Reproduction and Fertility

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    Stroma gene expression analyses. Values are shown as ∆∆ct, error bars are shown as s.e.m. *Significantly (P < 0.05) different values between the stimulation concentrations. n = 9 for every concentration including n = 3 biological replicates for each of the n = 3 SC donor animals.

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    Effects of embryonic sex on: (A) blastocyst size on days 8 and 10 and individual size increase during 48 h co-culture; (B) embryonic gene expression of IFNT, MX2, ISG15 and IFNAR1 on day 10 (qPCR data are shown as 15-∆Ct); (C) IFNT concentration measured in the cell culture supernatant after 48 h of co-culture with day 8 blastocysts and (D) endometrial stroma cell gene expression of MX2, OAS1, ISG15 and IFNAR1 after 48 h co-culture with day 8 blastocysts (qPCR data are shown as ∆∆Ct). Data of n = 26 female and n = 26 male embryo co-cultures are shown. The 25th percentile and the 75th percentile as well as the median are represented in the box plots. The whiskers correspond to the 95% CI of the data. Points represent outliers while a ‘+’ represents the extreme outliers. Significances are indicated as *P < 0.05, **P < 0.001.

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