Maternal-fetal cholesterol transport in the second half of mouse pregnancy does not involve LDL receptor-related protein 2.

AIM: LDL receptor-related protein type 2 (LRP2) is highly expressed on both yolk sac and placenta. Mutations in the corresponding gene are associated with severe birth defects in humans, known as Donnai-Barrow syndrome. We here characterized the contribution of LRP2 and maternal plasma cholesterol availability to maternal-fetal cholesterol transport and fetal cholesterol levels in utero in mice. METHODS: Lrp2+/- mice were mated heterozygously to yield fetuses of all three genotypes. Half of the dams received a 0.5% probucol-enriched diet during gestation to decrease maternal HDL cholesterol. At E13.5, the dams received an injection of D7-labelled cholesterol and were provided with 1-13 C acetate-supplemented drinking water. At E16.5, fetal tissues were collected and maternal cholesterol transport and fetal synthesis quantified by isotope enrichments in fetal tissues by GC-MS. RESULTS: The Lrp2 genotype did not influence maternal-fetal cholesterol transport and fetal cholesterol. However, lowering of maternal plasma cholesterol levels by probucol significantly reduced maternal-fetal cholesterol transport. In the fetal liver, this was associated with increased cholesterol synthesis rates. No indications were found for an interaction between the Lrp2 genotype and maternal probucol treatment. CONCLUSION: Maternal-fetal cholesterol transport and endogenous fetal cholesterol synthesis depend on maternal cholesterol concentrations but do not involve LRP2 in the second half of murine pregnancy. Our results suggest that the mouse fetus can compensate for decreased maternal cholesterol levels. It remains a relevant question how the delicate system of cholesterol transport and synthesis is regulated in the human fetus and placenta.

Characterization of relaxin binding in the uterus of the marmoset monkey.

The ovarian peptide hormone relaxin (RLX) plays an important role in the regulation of the endometrium both during the cycle and in early pregnancy. RLX interacts with specific receptors on endometrial stromal cells causing these to decidualize. In order to characterize the molecules with which RLX interacts in the primate uterus, a methodology based on a fully bioactive preparation of biotinylated porcine RLX was applied to cryosections of the uterus of female marmoset monkeys. Specific RLX binding was weakly detected in the proliferative phase in isolated endometrial stromal cells. In the secretory phase, the positively reacting cells increased in staining intensity and in number and also included some epithelial cells. Further increases occurred in pregnancy, but RLX binding in the endometrium decreased at the end of the cycle if pregnancy did not occur. The myometrium showed weak staining which did not vary through the cycle, but increased in pregnancy. Electrophoretic analysis of the RLX-binding moieties in these tissue sections indicated that a protein of approximately 40 kDa was the principal RLX-binding molecule, while minor specific bands were detectable at approximately 100 and approximately 200 kDa. The binding of biotinylated RLX could be specifically suppressed by co-incubation with unlabelled RLX, but not by insulin, IGF-I or biotin. This technique therefore allows the detection and molecular characterization of specific RLX binding in the primate uterus. In the marmoset monkey, the pattern of specific binding closely reflects the RLX-dependent physiology during implantation and early pregnancy, implying the probable involvement of a specific RLX receptor.

A local oxytocin system is part of the luteinization process in the preovulatory follicle of the marmoset monkey (Callithrix jacchus).

Luteinization is a complex differentiation process involving the interaction of extrinsic and intraovarian factors. The aim of this study was to examine the components of an intraovarian oxytocin (OT) system during the periovulatory period in the marmoset monkey, as well as the possible relationship of these components to other factors involved in the luteinization process, using immunohistochemistry and cell culture techniques. Ovaries were collected on Day 7 of the follicular phase (before the endogenous LH surge) and on Day 8 (22 h after an exogenous hCG application, but before ovulation). Before the endogenous LH increase, OT immunoreactivity was detectable at low levels in most antral follicles, where its presence was confined to antral granulosa cell (GC) layers. In contrast, immunoreactivity for the OT receptor (OTR) was localized primarily in the basal GC layer. After application of exogenous hCG, there was a marked enhancement in both the staining intensity and the number of cells positive for OT and the OTR in all GC layers of antral follicles, especially in the preovulatory follicle. Progesterone receptor and 3beta-hydroxysteroid dehydrogenase activity in GC were clearly present only when follicles were obtained after gonadotropin stimulation. Secretion of authentic OT was demonstrated from cultured GC obtained before the LH surge, with highest amounts in cells cultured from preovulatory as opposed to smaller antral follicles. OT production could be stimulated by the application of hCG to the GC cultured from preovulatory follicles, whereas the gonadotropin was without effect on GC from small follicles. FSH had no effect on OT production by GC from either follicle type. Application of OT to the cultures caused an increase in progesterone production by GC from large preovulatory follicles but was without effect on steroidogenesis by cells from small antral follicles. These results describing the presence and distribution of OT and OTR and their modulation by hCG, as well as the luteotrophic effect of OT in cultured GC from preovulatory follicles, implicate OT as a paracrine mediator in the luteinization process in the primate ovary.

Alpha 2-adrenergic binding sites in the medulla oblongata of tree shrews demonstrated by in vitro autoradiography: species related differences in comparison to the rat.

Alpha 2-adrenergic binding sites in the medulla oblongata of tree shrews and rats were detected and quantified by in vitro-autoradiography with the alpha 2-antagonist 3H-rauwolscine (3H-RAUW). The autoradiographic pattern of the radioligand binding in the tree shrew medulla oblongata resembles that which has been described by others for the human myelencephalon. This pattern coincides well with the occurrence of catecholaminergic structures detected by immunocytochemistry with antibodies against phenylethanolamine-N-methyltransferase and tyrosine hydroxylase. In contrast to the rat, where only the nucleus tractus solitarii and the nucleus dorsalis nervi vagi were labeled, five discrete nuclei specifically bound 3H-RAUW in tree shrews. The highest number of binding sites was detected in the nucleus dorsalis nervi vagi (nX; Bmax: 333 fmoles/mg) and the nucleus tractus solitarii (NTS; 311 fmoles/mg), followed by the nucleus nervi hypoglossi (nXII; 297 fmoles/mg), the nucleus reticularis parvocellularis (FRS; 230 fmoles/mg), and the area of the catecholamine cell groups A1 and C1 (area C1; 202 fmoles/mg). Maximal binding in the two labeled nuclei of the rat was 158 fmoles/mg. The discrete nuclei of the two species also showed different affinities for 3H-RAUW with Kd ranging from 0.17 to 0.83 nM in tree shrews and 1.80 to 1.95 nM in rats. Competition experiments revealed that the radioligand bound specifically to alpha 2-binding sites. In the tree shrew, nX, nXII and the area C1, also have a relatively high affinity for the alpha 1-antagonist prazosin which is a quality of the adrenoceptor subtype alpha 2B. Furthermore, in the area C1, 3H-RAUW binding was inhibited by the dopamine antagonist haloperidol. There are thus species related as well as regional differences with respect to the number, the affinity, and the pharmacological properties of alpha 2-binding sites in the medulla oblongata. In tree shrews, alpha 2-adrenoceptors can be autoradiographically quantified in regions which are not labeled in the rat, although former data predicted the existence of such receptors, e.g., in the area of the adrenaline cell group C1.