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 explant’s 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 mare’s 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 (1–2%) 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 17α-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 17β-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 17β-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, 17β-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

Oviductal and uterine leiomyomata in mares

This paper describes a case of a sessile uterine leiomyoma in a 17-year-old chronic infertile Selle Francais mare. The mass was removed by transendoscopic electrocoagulation. In the same period, 725 mares were screened for oviductal and uterine solid masses in a slaughterhouse survey. Two uterine masses and one oviductal mass were detected in three different mares. Histological and immunohistochemical examination revealed leiomyoma in the four masses. To the authors' knowledge, this is the first report of an oviductal leiomyoma in a mare

Fertiliteitsbehandelingen bij het paard: toepassingsmogelijkheden en beperkingen

Recent developments in the assisted reproduction in horses allow to breed foals from sub- and infertile mares, as well as from recently deceased mares or stallions. Oocytes can be obtained from live donor mares by ovum pick-up (OPU), by flushing oocytes from follicles using a transvaginal or transabdominal approach. Post mortem oocytes can be obtained by scraping the follicles. After oocyte maturation, the oocytes can be fertilized in vitro or can be transferred to the oviduct of an inseminated recipient mare (in vivo). Since conventional in vitro fertilization (IVF) is very unsuccessful in the horse, fertilization is performed by intracytoplasmic sperm injection (ICSI). After ICSI, the fertilized oocytes can be transferred to the oviduct of a synchronized recipient mare or further cultured in vitro up to the blastocyst stage. Subsequently, obtained blastocyts can be transferred to the uterus of a recipient mare. In this article, in vitro embryo production in the horse is highlighted, and possible advantages and disadvantages and clinical and scientific applications are reviewed

Update on mammalian sperm capacitation : how much does the horse differ from other species?

In contrast to various other mammalian species, conventional in vitro fertilization (IVF) with horse gametes is not reliably successful. In particular, stallion spermatozoa fails to penetrate the zona pellucida, most likely due to incomplete activation of stallion spermatozoa (capacitation) under in vitro conditions. In other mammalian species, specific capacitation triggers have been described; unfortunately, none of these is able to induce full capacitation in stallion spermatozoa. Nevertheless, knowledge of capacitation pathways and their molecular triggers might improve our understanding of capacitation-related events observed in stallion sperm. When sperm cells are exposed to appropriate capacitation triggers, several molecular and biochemical changes should be induced in the sperm plasma membrane and cytoplasm. At the level of the sperm plasma membrane, (1) an increase in membrane fluidity, (2) cholesterol depletion and (3) lipid raft aggregation should occur consecutively; the cytoplasmic changes consist of protein tyrosine phosphorylation and elevated pH, cAMP and Ca2+ concentrations. These capacitation-related events enable the switch from progressive to hyperactivated motility of the sperm cells, and the induction of the acrosome reaction. These final capacitation triggers are indispensable for sperm cells to migrate through the viscous oviductal environment, penetrate the cumulus cells and zona pellucida and, finally, fuse with the oolemma. This review will focus on molecular aspects of sperm capacitation and known triggers in various mammalian species. Similarities and differences with the horse will be highlighted to improve our understanding of equine sperm capacitation/fertilizing events

Embryotransplantatie bij het paard: onmisbaar in de moderne fokkerij

Nowadays, a valuable competition mare can produce offspring without interrupting its sport career which is made possible by a technique called embryo transfer. The valuable mare is inseminated and an embryo is flushed seven days later. The early embryo is then transferred to the uterus of a recipient mare that carries the pregnancy to term. In 50% of the cases, flushing of the donor mare results in an embryo. After transfer, an average of 70% of the recipient mares become pregnant. These percentages are influenced by several factors related to both the donor and recipient mares

Progress in biomedication using live foods

With the intensification of aquaculture production systems, mass mortality of fish and shellfish caused by bacterial infections is not unusual. Chemotherapy (treatment of infection with antimicrobial compounds) and immunoprophylaxis (vaccination and immunostimulation), are the common methods applied to treat bacterial infections. In search of an efficient and cost-effective delivery route for antimicrobial agents and/or immuno-prophylactics to treat fish and shrimp larvae, the use of live prey organisms bioencapsulated with drugs has been explored. This article provides an update on the use of the bioencapsulation technique in aquaculture