No abstract

Previously, we have shown that the O4 antibody can be used to define and purify olfactory nerve ensheathing cells (ONECs) from the rat olfactory bulb by fluorescence-activated cell sorting. In this study, using a larger panel of neural markers, we demonstrate that this apparently homogeneous population of ONECs possess a heterogeneous antigenic profile both in vivo and in vitro. The antigenic profile of the sorted cells initially correlated with their antigenic profile in vivo, although expression of some of the markers was either lost or gained during time in culture. These changes were influenced by the culture conditions, with a greater loss of "typical" ONEC markers in serum-containing medium. In serum-free medium, which maintains the cells in a phenotype that closely resembles their in vivo counterparts, we were able to reclassify the ONECs into two cell types based on morphology and antigenic phenotype by using antibodies to polysialic acid (correlating with the embryonic form of N-CAM expression) and the low-affinity nerve growth factor receptor. A detailed immunocytochemical study of the developing olfactory system showed that these two cell types could also be detected along the entire length of the olfactory nerve and the outer layer of the olfactory bulb from Embryonic Day 14 to adulthood, suggesting they were not an in vitro artefact. To address the relationship between the two cell types we constructed a clonal ONEC cell line by retroviral infection with the temperature-sensitive mutant gene of the large T antigen. This clonal cell line contained cells that expressed antigenic phenotypes of both classes of ONECs, suggesting that both cell types are related and share a common lineage.

The G protein-coupled receptor GPR54 (AXOR12, OT7T175) is central to acquisition of reproductive competency in mammals. Peptide ligands (kisspeptins) for this receptor are encoded by the Kiss1 gene, and administration of exogenous kisspeptins stimulates hypothalamic gonadotropin-releasing hormone (GnRH) release in several species, including humans. To establish that kisspeptins are the authentic agonists of GPR54 in vivo and to determine whether these ligands have additional physiological functions we have generated mice with a targeted disruption of the Kiss1 gene. Kiss1-null mice are viable and healthy with no apparent abnormalities but fail to undergo sexual maturation. Mutant female mice do not progress through the estrous cycle, have thread-like uteri and small ovaries, and do not produce mature Graffian follicles. Mutant males have small testes, and spermatogenesis arrests mainly at the early haploid spermatid stage. Both sexes have low circulating gonadotropin (luteinizing hormone and follicle-stimulating hormone) and sex steroid (␤-estradiol or testosterone) hormone levels. Migration of GnRH neurons into the hypothalamus appears normal with appropriate axonal connections to the median eminence and total GnRH content. The hypothalamicpituitary axis is functional in these mice as shown by robust luteinizing hormone secretion after peripheral administration of kisspeptin. The virtually identical phenotype of Gpr54-and Kiss1-null mice provides direct proof that kisspeptins are the true physiological ligand for the GPR54 receptor in vivo. Kiss1 also does not seem to play a vital role in any other physiological processes other than activation of the hypothalamic-pituitary-gonadal axis, and loss of Kiss1 cannot be overcome by compensatory mechanisms.Gpr54 ͉ kisspeptin ͉ mouse ͉ puberty N euroendocrine events within the hypothalamus control sexual maturation and seasonal breeding in mammals (1). The gonadotropin-releasing hormone (GnRH)-induced secretion of the gonadotropic hormones luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary is essential to invoke puberty and maintain reproductive function. The G protein-coupled receptor GPR54 (2) is a key protein involved in the pubertal activation of the hypothalamic pituitary gonadal axis because mice and humans with mutations in this receptor are sterile with hypogonadotropic hypogonadism (3-7).A series of overlapping peptide ligands (kisspeptins) for the GPR54 receptor are produced by the Kiss1 gene (8-10). Kiss1 mRNA is expressed in hypothalamic regions that regulate gonadotropin secretion including the anteroventral periventricular nucleus (AVPV), the periventricular nucleus, and the arcuate nucleus (ARC) (11). Kiss1 expression increases at puberty in rodents (12-14) and primates (15) and fluctuates during the rat estrous cycle (12,16). Kiss1 expression is also subject to differential regulation by sex steroids, providing a plausible mechanism for the feedback control of gonadotropin secretion by these hormones (17, 18). Ad...