Interaction off αT3-1 Cells with Lactotropes and Somatotropes of Normal Pituitary in vitro

Abstract

A pituitary cell population of 14-day-old female rats, containing lactotropes and somatotropes but deprived of gonadotropes, was prepared by unit gravity sedimentation through a serum albumin gradient and allowed to reaggregate either as such or after mixing this population with cells of the gonadotropic αT3-1 cell line. In a perifusion system gonadotropin-releasing hormone (GnRH) had no effect on prolactin (PRL) and growth hormone (GH) release in the former aggregates but stimulated PRL release in the latter. In the αT3-l cell-containing aggregates GnRH showed a biphasic effect on GH release: inhibition during exposure to GnRH followed by a rebound secretion upon removal of the peptide. The aggregation capacity of αT3-l cells with the normal pituitary cells was demonstrated by using an αT3-l cell clone stably transfected with the reporter gene β-galactosidase. Perifusion of the gonado-trope-deprived aggregates with medium conditioned by αT3-1 cell provoked a rapid stimulation of PRL release and a biphasic effect on GH release. Medium conditioned by the cortico-tropic cell line AtT20 also stimulated PRL release but had no concomitant effect on GH release. Medium conditioned by αT3-l cells, when added for 40 h to aggregates of 14-day-old rat pituitary, provoked an increase in the number of ³H-thymidine (³H-T)-labelled lactotropes and a decreased in the number of ³H-T-labelled somatotropes. The conditioned medium was concentrated on Sep-Pak C18 and ultrafiltrated through an Amicon membrane with 3-kD molecular weight cut-off and the retained molecules separated by reversed-phase HPLC. The material stimulating ³H-T labelling of lactotropes eluted from the column with a different retention time than material inhibiting ³H-T labelling of somatotropes, suggesting that the effect on lactotropes is mediated by (a) molecule(s) different from that affecting somatotropes. The effects of αT3-l cells and their secretion products on lactotropes and somatotropes were comparable to those we previously observed using enriched populations of normal gonadotropes. The HPLC elution profiles of the substances affecting ³H-T incorporation as well as the specificity of these effects were also similar to that of the substances isolated previously from gonadotrope-conditioned medium. The present data, therefore, support previous conclusions on the paracrine control of the lactotrope/somatotrope lineage by the gonadotropes. Further purification and chemical characterization of the growth factors with selective action on lactotropes and somatotropes may lead to a better understanding of the development of the latter lineage.

Anterior pituitary cell networks

“…Based upon morphological observations, as well as findings from re-aggregation studies, an important role for cell-cell communication for pituitary function and hormone release has long been suspected. For example, cup-shaped lactotrophs embrace and surround gonadotrophs in multiple species (Gregory et al, 2000;Hovarth et al, 1977;Tortonese et al, 1998), gonadotroph-conditioned medium evokes PRL secretion from cultured lactotrophs (Andries et al, 1995;Denef and Andries, 1983), application of a gonadotroph-specific secretagogue (GnRH) upregulates lactotroph output (Henderson et al, 2008), and gap junction-signaling is positively correlated with circulating PRL levels (Vitale et al, 2001). Whilst it is beyond the scope of the current review to discuss intrapituitary communication in detail (please refer to recent reviews from ours and other groups (Denef, 2008;Hodson et al, 2012a)), the important routes will nonetheless be highlighted within the context of endocrine cell networks.…”

Estrogen receptor signaling is an unstable feature of the gonadotropic LβT2 cell line

“…In females, E2 exerts a negative feedback on LH release for the greater part of the ovarian cycle, at least in part through direct actions at the pituitary level. This inhibitory action is transiently reversed into marked facilitation of pituitary LH secretion during the mid-cycle LH surge (Knobil, 1988;Herbison, 1998;Kerdelhué et al, 2002;Moenter et al, 2003) The role of E2 in the GnRH-mediated regulation of LH release by pituitary gonadotrophs has been extensively investigated using primary pituitary cell cultures and isolated perifused pituitaries (Emons et al, 1989;Ortmann et al, 1992a;Ortmann et al, 1992b) However, gonadotrophs represent only 10-15% of the anterior pituitary cell population (Wang, 1988) It has been established that paracrine interactions between gonadotrophs and other pituitary cell types, including lactotrophs and somatotrophs, may influence basal or GnRH-induced LH secretion (Cheung, 1983;Denef and Andries, 1983;Andries et al, 1995;Gregory et al, 2004), hereby complicating data interpretation. The need for in vitro models to study estrogenic effects at the pituitary level is further emphasized by the increasing interest for the therapeutic use of phytoestrogens and the development of selective estrogen receptor modulators on the one hand and growing concern for potentially disrupting effects of chemical pollutants with estrogenic actions on reproductive function on the other hand (Eertmans et al, 2003) A c c e p t e d M a n u s c r i p t…”

Endogenous Cannabinoids: Structure and Metabolism

“…The paracrine cross-talk with lactotrophs and somatotrophs was also confirmed by coaggregation of a lactotrophs/somatotroph population from 14-day-old rats with cells of the gonadotrophic cell line αT3-1 (101). It was found that GnRH induced a stimulation of PRL release and a dual effect on GH release, although the magnitude of the response was smaller than in normal pituitary.…”

“…Mediation by paracrine factor(s) was indicated by the finding that medium conditioned by a highly enriched population of gonadotrophs cultured in the presence of GnRH, mimicked the effects of GnRH (120, 121). Also, medium conditioned by the gonadotroph precursor cell line αT3-1 contained material stimulating development of lactotrophs and inhibiting development of somatotrophs (101). Four substances with a selective mitogenic effect on lactotrophs were partially purified by high-performance liquid chromatography fractionation, whereas two other substances were isolated with antisomatotroph action (120, 121).…”