Deciphering the embryo-maternal dialogue in the horse using an oviduct explant model

To date, in vivo derived equine embryos are of superior quality compared to those produced in vitro, in terms of morphology and ultrastructure, gene expression and developmental competence. This phenomenon confirms that current in vitro systems are deprived of particular essential maternal factors or signals and illustrates the importance of embryo-maternal interplay. So far, only very few signals involved in the embryo-maternal dialogue have been identified in the horse. Although in vivo models are the gold standard, it is difficult to investigate molecular processes within a relatively large space like the equine oviduct. It is for instance difficult to locate gametes and the embryo in the oviduct and to unravel local paracrine and autocrine events, which are essential in elucidating intra- and extracellular molecular pathways and processes. The general goal of this thesis was to gain insight in the embryo-maternal communication in the horse. To achieve this aim, the influence of steroids on the oviduct was in vivo investigated (CHAPTER 4). These effects were simulated in vitro (CHAPTER 5.1) by means of an optimized in vitro oviduct explant model (CHAPTER 3). The effect of steroids (CHAPTER 5.1) and embryos (CHAPTER 5.2) on oviduct explantU+2019s gene expression was assessed. Next, as a step forward to the improvement of in vitro equine embryo culture, it was also investigated whether embryos in vitro benefit from culture at the mareU+2019s body temperature (CHAPTER 6). In CHAPTER 3.1, a culture system which sustains equine oviduct explants bordered by highly differentiated, glucose consuming, functional and intact epithelial cells showing vigorous ciliary activity during 6 days of culture was optimized. Only a negligible percentage (1U+20132%) of the cells in the explants showed features of apoptosis or necrosis and therefore, it could be concluded that the explants mimic the in vivo situation very closely. Although dark-cell degeneration, a hypoxia related type of cell death, was detected using TEM, the hypoxia marker genes HIF1A, GLUT1 and VEGFA were not upregulated in the culture system, even in culture at high oxygen concentrations. In an attempt to further unravel the origin of the dark cell degeneration, explants were cultured with and without foetal calf serum (FCS) or supplemented with serum replacement insulin-transferrin-selenium (ITS) in CHAPTER 3.2. It turned out that selenium increases while FCS decreases to some extent the incidence of explants showing dark cell degeneration. In the horse no information is available concerning local steroid concentrations in the oviduct and their fluctuations during the oestrous cycle. Therefore in CHAPTER 4 the concentrations of progesterone, oestradiol, testosterone and 17-hydroxyprogesterone were determined in equine oviductal tissue by the highly powerful technique UHPLC-MS/MS whereas RIA was applied to measure steroids in oviductal fluid. Progesterone concentrations were high in oviductal tissue and fluid ipsilateral to the ovulation side during diestrus, whereas testosterone, and 17U+03B1-testosterone and 17-hydroxyprogesterone other steroid hormone concentrations were not influenced by the side of ovulation. The most plausible explanation for the elevated progesterone concentration in the ipsilateral oviduct of the mare is a combination of 1) the contribution from follicular fluid in the oviduct and the diffusion of follicular fluid steroids after ovulation; 2) a local transfer of steroids via blood or lymph, 3) local synthesis of progesterone in the oviduct which is confirmed by the expression of StAR, cytochrome P450scc and 3-beta-HSD, key enzymes in the progesterone synthesis, as well as aromatase, which is suggestive of local steroidogenesis; and, 4) the paracrine contribution from follicular cells. In CHAPTER 5.1, preovulatory explants were stimulated with hormone concentrations as they prevail in the postovulatory stage and vice versa. The influence of these steroid hormones on the function (ciliary activity, glucose consumption and lactate production), the ultrastructure, the mRNA expression of a set of embryotrophic genes, the steroidogenic capacities and the progesterone receptor expression in the equine oviduct was assessed. Progesterone and 17U+03B2-oestradiol were able to modify ciliary activity, energy metabolism, gene expression, immunoreactive steroidogenic enzyme expression and progesterone receptor expression in oviductal explants in vitro. Furthermore, PAI1, PLAU, GLUT1, CSF1, TGFA and MMP2 were shown to be upregulated in the oviductal epithelium originating from mares in the postovulatory cycle stage. Moreover, preovulatory oviduct explants, primed by steroids in vivo, are responsive to in vitro stimulation with postovulatory oviductal progesterone and 17U+03B2-oestradiol concentrations and approach the in vivo condition at the level of functionality and gene expression. This endorses that our explant model remains functional and responsive for at least three days (CHAPTER 3). In addition, it turned out that oviduct explants are capable of producing large amounts of progesterone in vitro and are able to remove considerable amounts of oestrone, 17U+03B2-oestradiol and testosterone from the culture medium. This confirms again the functional integrity of the culture system. The oviductal environment represents the optimal environment for early embryo development. Supposing that oviductal cells provide specific mitogenic factors that would normally be present in the oviduct, or non-specific factors that improve the culture environment such as reduction of oxygen tension, removal of waste products or provision of substrates and co-factors, we cultured equine zygotes, obtained by ICSI, with and without equine oviduct explants in CHAPTER 5.2. To elucidate the role of developing embryos on the modulation of gene expression in the oviduct, we unraveled the response of the same set of embryotrophic genes as used in CHAPTER 5.1 in oviductal cells cultured together with equine putative zygotes (CHAPTER 3). Co-culture with equine embryos stimulated the expression of the embryotrophic genes TIMP1, PTGER2, TGFA, MMP2, CSF1 and PAI1 in the oviduct explant, which have been described to be involved in embryo transport, and in stimulating embryonic development and quality, and they modulate oviductal matrix turnover. Co-culture did not affect ciliary activity or viability of oviduct explants. In an attempt to further improve embryo culture conditions, it was investigated in CHAPTER 6 if oocyte maturation, cleavage, blastocyst rate and blastocyst diameter could be improved when applying the physiological body temperature of the mare (37.3 °C) rather than the conventional 38.5°C. Cytoplasmic maturation does not differ in both groups. The size of blastocysts was smaller in the oocytes matured and embryos cultured at 37.3°C compared with the matured and cultured at 38.5°C. Since blastocyst size is a parameter of embryo viability, culture at 38.5°C may be recommended rather than culture at 37.3°C. In the final CHAPTER 7 the general discussion and conclusions are presented. Our findings indisputably demonstrate that the equine oviduct is able to respond to both steroids and embryonic signals in vivo and in vitro and that our oviduct explant model is an excellent model to further unravel the embryo-maternal interplay in the horse.

ISBN:9789058644404

Jaar van publicatie:2015

Toegankelijkheid:Open