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Steroids play an important role in mammalian reproduction and early pregnancy. Although systemic changes in steroid concentrations have been well documented, it is not clear how these correlate with local steroid concentrations in the genital tract. We hypothesised that, in the horse, the preimplantation embryo may be subjected to high local steroid concentrations for several days. Therefore, we measured progesterone, 17-hydroxyprogesterone, 17β-oestradiol, testosterone and 17α-testosterone concentrations in equine oviductal tissue by ultra-HPLC coupled with tandem mass spectrometry, and progesterone, 17β-oestradiol, oestrone and testosterone concentrations in oviduct fluid by radioimmunoassay, with reference to cycle stage and side of ovulation. Progesterone concentrations were high in oviductal tissue and fluid ipsilateral to the ovulation side during dioestrus, whereas other steroid hormone concentrations were not influenced by the side of ovulation. These results suggest that the high ipsilateral progesterone concentration is caused by: (1) contributions from the follicular fluid in the oviduct and diffusion of follicular fluid steroids after ovulation; (2) local transfer of steroids via blood or lymph; (3) local synthesis of progesterone in the oviduct, as evidenced by the expression of steroidogenic enzymes; and (4) a paracrine contribution from follicular cells. These data provide a basis for the study of the importance of endocrine and paracrine signalling during early embryonic development in the horse.

The oviduct undergoes dramatic functional and morphological changes throughout the oestrous cycle of the mare. To unravel the effects of steroids on the morphology, functionality and gene expression of the equine oviduct, an in vitro oviduct explant culture system was stimulated with physiological concentrations of progesterone and 17β-oestradiol. Four conditions were compared: unsupplemented preovulatory explants, preovulatory explants that were stimulated with postovulatory hormone concentrations, unsupplemented postovulatory explants and postovulatory explants that were stimulated with preovulatory hormone concentrations. The modulating effects of both steroids on oviduct explants were investigated and the following parameters examined: (1) ciliary activity, (2) glucose consumption and lactate production pattern, (3) ultrastructure, (4) mRNA expression of embryotrophic genes, (5) steroidogenic capacities of oviductal explants and (6) progesterone receptor expression. The present paper shows that the equine oviduct is an organ with potential steroidogenic capacities, which is highly responsive to local changes in progesterone and 17β-oestradiol concentrations at the level of morphology, functionality and gene expression of the oviduct. These data provide a basis to study the importance of endocrine and paracrine signalling during early embryonic development in the horse.

Isolation of pure inner cell mass (ICM) and trophoblast samples from a single blastocyst is necessary to obtain accurate information on the transcriptome of these cells. Microsurgical techniques have been described to separate the ICM and trophoblast, but unfortunately, contamination of the ICM cell population with trophoblastic cells is inevitable with these methods. Alternatively, immunosurgery has been described as a valuable technique to obtain a pure ICM sample, although this technique seems to alter the normal gene expression pattern. Laser capture microdissection (LCM) provides the possibility of isolating small tissue fractions from heterogeneous tissue sections, without contamination by the surrounding tissue and without changing the gene expression pattern of the cells. In this study, a protocol is described for the application of LCM to isolate homogeneous ICM and trophoblast samples from single bovine blastocysts for downstream gene expression analysis. The absence of contaminating trophoblastic fractions in the isolated ICM cells was controlled with primers for the keratin 18 (KRT18) gene, which is considered a trophoblast-specific marker in bovine blastocysts. Expanded blastocysts were produced by routine in vitro methods described by (Vandaele et al. 2010 Reproduction 139, 505–511) and fixed in a modified methacarn solution for 24 h. After fixation, the blastocysts were embedded in RNase-free soluble agarose 2%, processed in an STP 420D Tissue Processor, embedded in paraffin, cut in serial sections, and adhered to glass slides, followed by deparaffinization in xylene and staining of the sections with 0.1% cresyl violet in a 85% ethanol solution. Laser capture microdissection was performed as described previously by (De Spiegelaere et al. 2008 Anal. Biochem. 382, 72–74). The ICM was isolated by placing the same cap over 3 to 4 serial sections of one blastocyst. Subsequently, the same procedure was performed with a second cap to isolate the trophoblast. Total RNA was isolated from the LCM-derived ICM and trophoblast on the caps and converted into cDNA. Gene-specific primers for KRT18 (5′-GCAGACCGCTGAGATAGGA-3′ and 5′-GCATATCGGGCCTCCACTT-3′) and for 18S rRNA, a commonly used reference gene (5′-AGAAACGGCTACCACATCCA-3′ and 5′-CACCAGACTTGCCCTCCA-3′), were used and PCR was carried out. Expression of the control gene 18S rRNA was readily detectable in all cell samples. Keratin 18 was detectable in LCM-derived trophoblast, but was absent in the LCM-derived ICM cells, indicative of the successful isolation of ICM cells without contaminating trophoblastic cells. This study demonstrates a novel approach for the application of LCM on small tissue samples that are difficult to handle and which can be used for molecular analysis of specific cell lineages within embryos of different species. Supported by the Fund for Scientific Research–Flanders, Belgium, aspirant 1.1.477.07N00.