Embryonic development and uterine influence in the pig

In this thesis, next to morphological examinations of the porcine embryoblast in the second week of gestation, several parameters to study porcine embryogenesis, especially gastrulation and the development of the anterior-posterior axis, were developed. The use of molecular markers as parameters allowed a more detailed description of these processes in the porcine embryoblast. From our results, it can be concluded that the expression of the molecular markers gsc en ActRII is evolutionary conserved between pig, chicken and mouse in early embryonic development. Moreover, using gsc expression, it was shown that the earliest development of the anterior-posterior axis was visualized by movement of the lower layer of the embryoblast and was similar to the processes of hypoblast movement inchicken and AVE movement in mouse. Using tracers during in vitro culture of porcine embryoblasts would be a valuable tool to study the movement of cells in the embryo, gaining more knowledge about the formation of the endoderm layer and the other germ layers. Since the expression patterns of essential genes appear evolutionary conserved, these genes could also be used as parameters for quality control of in vitro fertilized (IVF) and produced (IVP) embryos, not only in the pig, but also in other domestic ungulates such as the cow. In this respect, no molecular markers have been developed yet to qualify the human embryo after in intracytoplasmic sperm injection (ICSI), but only a morphological control is performed (Miller and Smith, 2001). The use of molecular markers would surely be beneficial to improve these artificial methods to generate embryos. The final goal of the thesis was to study a possible influence of porcine uterine activin on embryonic development, supposed to be important during the period of high embryonic mortality in the second week, when also mesoderm induction and anterior-posterior axis development take place. Indeed, we found high concentrations of activin in the uterine flushings, while the activin receptor was present in the embryoblast during these stages. Moreover, expression of the antagonist for activin, follistatin, was not detected in the uterus in these stages of development. A direct experimental model, like in vitro culture of the embryos, to test the hypothesis that uterine factors, such as activin, might influence embryogenesis has not been developed yet. Also, our own attempts to culture porcine embryos failed. Another way to examine the influence of the uterus would be by administration of progesterone, since this has been suggested to induce a change in the uterine secretional pattern. However, we did not find differences in activin or total protein levels in the uterine flushings between the progesterone treated and the control group at slaughter at day 11 after estimated ovulation. The observation that embryonic survival was higher because of the survival of the less developed embryos only in the progesterone treated group, while the progesterone receptor in the embryo is present only before the morula stage, suggests that progesterone might influence the uterine secretions. Three possible explanations of this influence on uterine secretions and higher embryonic survival rates are: 1) a uterine factor is still secreted that is required for the less developed embryos to continue their development, 2) a uterine factor is secreted that rescues the less developed embryos from a hostile uterine environment induced by the more developed littermates, 3) the effect of oestradiol, synthesized by the more developed littermates, on the uterus is reduced and therefore the sudden change in the uterine environment does not take place. Re-examination of the uterine flushing fluids from our experiments on several other (growth) factors, such as FGF's, IGF's or TGFß's, could provide more clues on the mechanisms of survival of the less developed littermates. The lack of an in vitro culture system for porcine embryos was and still is a serious drawback. The advantages of such system would be two-fold. Firstly, as mentioned above, tracer studies to map and assess cell movement during gastrulation and the development of the anterior-posterior axis would provide more insight in these processes, next to the use of more molecular markers. Secondly, it would become possible to directly examine the influence of specific factors, possibly of maternal origin, on specific developmental processes in the porcine embryo. Therefore, the development of such a system for the porcine embryoblast should have a high priority in the future

Uterine-embryonic interaction in pit : activin, follistatin, and activin receptor II in uterus and embryo during early gestation

The mRNA expression patterns of activin A and follistatin in the uterus and embryo, the mRNA expression of the activin receptor II in the embryo, and the localization in the uterus of the immunoreactive activin A and the receptor II proteins in the uterus were examined at gestation days 7-12 after ovulation in pig. Activin was located predominantly at the mesometrial side of the uterus during all stages of pregnancy studied. Follistatin mRNA was absent in the uterus during these stages, suggesting that activin of uterine origin is not inhibited by intra-uterine follistatin. The receptor was localized throughout the glandular and luminal epithelium of the uterus. In the embryo, activin was expressed predominantly in the epiblast before unfolding, but after unfolding of the epiblast activin expression shifted to the trophoblast. The expression pattern of follistatin mRNA was contrarily to that of activin, i.e., before unfolding predominantly in the trophoblast (days 8-9), and shifted to the epiblast at day 10. During streak stages, follistatin was detected in the node and primitive streak. Activin receptor II mRNA was first detected at day 8 in the embryoblast. At day 11, it was expressed in trophoblast cells near the epiblast, and in the first ingressing mesoderm cells. During the streak stages, it was expressed predominantly in the trophoblast. The presence of activin and its receptor in uterine epithelium and early embryonic tissues indicate that both embryonic and uterine activin are involved in intra-uterine processes, such as attachment and early embryonic development

Chemokine CXCL13 is essential for lymph node initiation and is induced by retinoic acid and neuronal stimulation

The location of embryonic lymph node development is determined by the initial clustering of lymphoid tissue-inducer (LTi) cells. Here we demonstrate that both the chemokine CXCL13 and the chemokine CCL21 attracted LTi cells at embryonic days 12.5-14.5 and that initial clustering depended exclusively on CXCL13. Retinoic acid (RA) induced early CXCL13 expression in stromal organizer cells independently of lymphotoxin signaling. Notably, neurons adjacent to the lymph node anlagen expressed enzymes essential for RA synthesis. Furthermore, stimulation of parasymphathetic neural output in adults led to RA receptor (RAR)-dependent induction of CXCL13 in the gut. Therefore, our data show that the initiation of lymph node development is controlled by RA-mediated expression of CXCL13 and suggest that RA may be provided by adjacent neurons