75 EXPRESSION OF GROWTH FACTOR GENES IN IN VITRO-PRODUCED BLASTOCYST CHANGES AFTER UTERINE PASSAGE, BUT ENDOMETRIAL EXPRESSION IS UNAFFECTED BY THE PRESENCE OF EMBRYOS

Abstract

Growth factors (GFs) exert recognised roles in mammalian reproduction. However, little is known about the role of GFs in very early development. We examined here endometrial and embryonic transcription of genes coding for GF proteins identified in bovine uterine fluid (UF): artemin, stanniocalcin-1 (STC-1); heparin-binding EGF-like growth factor (HBEGF); connective tissue growth factor (CTGF); and stem cell factor (SCF). Embryos were in vitro produced (IVP) including culture in SOF+BSA. On Day 6, embryos (n = 50) were transferred into the uteri of oestrus-synchronized heifers (n = 10). On Day 8, embryos were flushed, and groups (n = 6) of expanded blastocysts (n = 10) were snap frozen in LN₂ and stored at –145°C. Day 8 embryos cultured in vitro from Day 6 were used as controls (n = 6 × 10 embryos). Transcript levels were also analysed in endometrial tissue collected from Day 8 slaughtered females that were embryo (n = 4) or sham transferred (n = 4). Samples were incubated overnight at 4°C in RNAlater and stored at –145°C in an ultrafreezer. Samples analysed (n = 3 caruncle; n = 3 intercaruncle) were taken from ipsilateral middle horn region. Embryonic and endometrial samples were analysed in duplicate for RT-qPCR analysis. Data were analysed by Wilcoxon Mann-Whitney test. In embryos, uterine passage compared with IVP down-regulated artemin (P < 0.01) and STC1 (P < 0.05) expression (0.61 ± 0.04 and 0.81 ± 0.06 v. 1.54 ± 0.10 and 1.25 ± 0.17, respectively). Conversely, abundance of CTGF (P < 0.05) and HBEGF (P < 0.01) decreased in IVP v. uterus-exposed embryos (0.79 ± 0.04 and 0.81 ± 0.03 v. 1.38 ± 0.18 and 1.37 ± 0.23, respectively). No changes were observed for SCF (0.96 ± 0.16 v. 1.09 ± 0.05 in IVP embryos). Endometrial gene expression for each GF did not change in response to embryos v. sham transfer (P > 0.10). Caruncular expression was higher (P < 0.05) for SCF compared with intercaruncles (1.32 ± 0.13 v. 0.88 ± 0.13). However, strong positive correlations (P < 0.006 to P < 0.0001) were seen for HBEGF with STC-1 (r = 0.67) and SCF (r = 0.72); for SCF with STC-1 (r = 0.61); and for CTGF with STC-1 (r = 0.59) and SCF (r = 0.50), suggesting a common transcription regulation among some GFs. Changes in embryonic gene expression reflect a regulatory response to the cognate GFs in the UF, which suggests a relevant role for GFs in early embryo-maternal interactions. Within the endometrium, our observations are consistent with studies postulating faster, non-transcriptional responses to early embryos (Gómez and Muñoz, Reproduction 2015 150, R35–R43). Alternatively, it is possible that very local endometrial responses for the analysed GFs were not detected despite the presence of multiple embryos.

Research was supported by MICINN, project AGL2012–37772, and FEDER. The authors are members of the COST Action FA1201 (Epigenetics and Periconception environment).