148 EXPRESSION OF ARTEMIN IN THE BOVINE ENDOMETRIUM, UTERINE FLUID, AND EARLY EMBRYOS

Abstract

Artemin, a member of the glial cell line-derived neurotrophic factor (GDNF) family, is expressed in human and mice pre-implantation embryos and reproductive tract (Li et al. 2009 FEBS Lett. 583; Kawamura et al. 2012 PLoS One 7). In mice, artemin promotes in vitro embryo development and decreases apoptosis (Li et al. 2009 FEBS Lett. 583). The presence of artemin in cattle embryos and endometrium, however, is unknown. In this work we analysed artemin expression in bovine blastocysts and endometrium by immunohistochemistry and in uterine fluid (UF) by Western blot (WB). Briefly, Day-6 in vitro-produced (IVP) embryos (n = 50) were nonsurgically transferred to the uterus of heifers (n = 10, 50 IVP embryos per heifer) at nonconsecutive oestrus cycles. On Day 8, embryos and their corresponding diluted UF were flushed; blastocysts that developed entirely in vitro were also collected. In addition, endometrial samples were collected on Day 8 from slaughtered females that were embryo transferred (n = 6) and sham transferred (n = 6) on Day 5. Artemin localization was investigated in blastocysts and in endometrial samples, using immunohistochemical staining methods described elsewhere (Muñoz et al. 2012 J. Proteome Res. 11; Gómez et al. 2014 Reproduction pii: REP-14–0304). The signal-strength comparisons between uterus-exposed and IVP blastocysts were analysed using the software Confocal Uniovi Image-J. Quantification of WB protein bands was achieved by computer-assisted densitometry using Image-J software. Artemin was detected, with similar intensity, in the inner cell mass and trophectoderm from both uterus-developed and IVP blastocyst. All embryos analysed expressed artemin. The signal intensity and staining pattern observed did not differ between uterus-exposed and IVP blastocysts. In the endometrium, the most intense staining for artemin was localised to the apical sites in the luminal epithelium and in the glandular epithelium of superficial glands. There was also diffuse staining in the stroma and deep uterine glands. The uterine region and pregnant or cyclic status did not affect the artemin staining pattern. Artemin was detected by WB in all UF samples analysed (embryo transferred N = 10, sham transferred N = 10). However reliable quantitation of artemin by WB was unfeasible due to the broad dynamic range of artemin expression through samples. In conclusion, our results demonstrate the presence of artemin in bovine uterine endometrium and UF, and embryos during early development. As shown in mice, it is feasible that artemin might exert an autocrine/paracrine role during early embryo development in the cow.

The study received grant support: Spanish Ministry of Science and Innovation (MINECO, project AGL2012–37772 and FEDER). M. M. was supported by grant MICINN-RYC08–03454. The authors are members of the COST Action FA1201 Epiconcept.