Heterologous Down-Modulation of Luteinizing Hormone Receptors by Prolactin: A Flow Cytometry Study*

Abstract

Although the binding, internalization, and regulation of LH, PRL, and their respective receptors have been extensively studied, it is not known whether the receptors are coordinately regulated. Using double labeling experiments, we have previously shown that receptor-bound LH and PRL can be colocalized in identical endosomes of granulosa cells. We hypothesize that high levels of PRL may induce a heterologous down-modulation of LH receptors, consequently reducing ovarian responsiveness to further gonadotropin stimulation. In this study we used a novel procedure to enrich endosomes containing internalized PRL and to determine whether unoccupied LH receptors were cointernalized in granulosa cells. Porcine granulosa cells were obtained from medium-sized (3-5 mm) follicles and cultured for 4 days in the presence of FSH. Fluorescein isothiocyanate-labeled PRL (FITC-PRL) was used as a ligand to induce internalization of PRL receptors and as a marker to label endosomes. Granulosa cells were incubated with FITC-PRL at either 4 or 37 C for various times. At the end of the incubation, cells were trypsinized to remove surface receptors and then homogenized. The postnuclear fraction containing endosomes and other subcellular organelles was sorted using a FACStar Plus cell sorter. Results from 14 separate sorting experiments showed that 1) FITC-PRL-treated cells exhibited a sorting pattern distinct from that of FITC-BSA-treated or untreated cells; 2) excess unlabeled PRL partially shifted the sorting profile to one similar to that in controls; 3) the differences in sorting profiles were not due to free FITC; and 4) using this method, it was possible to isolate FITC-PRL-containing endosomes that were virtually devoid of other contaminating subcellular particles. Fluorescently positive (FITC-PRL-containing) organelles were collected and assayed for LH receptors using [125I]hCG as a tracer. When the cells were incubated with FITC-PRL at 37 C for 3 h, the number of available LH receptors (as determined by [125I]hCG binding) was 37% higher in particles containing FITC-PRL than in those devoid of FITC-PRL. If the cells were allowed to preincubate with FITC-PRL at 4 C for 10-16 h before raising the temperature to 37 C, the number of available LH receptors in FITC-PRL-containing endosomes was about 7-fold higher than that in FITC-negative endosomes. Results from this study suggest that PRL not only induces internalization of its own receptor, but also causes down-modulation of unoccupied LH receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of Dopamine on the Altered Release of Prolactin, Luteinizing Hormone, and Follicle-Stimulating Hormone Induced by Interleukin-2 in vitro

“…Denef and Andries [26] reported the paracrine interaction between lacto trophs and gonadotrophs in pituitary cell aggregates. Lane and Chen [27] recently suggested that PRL, in addi tion to inducing the internalization of its own receptors, also caused a down-modulation of unoccupied LH recep tors, indicating a close link between PRL and LH cells. However, after incubation with medium containing high K+, only the release of PRL was inhibited by prior in cubation with DA, but the release of LH and FSH was unaltered which probably illustrates different mecha nisms operating before and after treatment with medium containing high K + .…”

Three Different Turkey Luteinizing Hormone Receptor (tLH-R) Isoforms II: Characterization of Differentially Regulated tLH-R Messenger Ribonucleic Acid Isoforms in the Ovary

“…Furthermore, it was shown experimentally that induced hyperprolactinemic rats demonstrated impaired ovarian function that was associated with a significant reduction in available LH-R [18,19]. Two different alternatively spliced forms of tLH-R (tLH-R insert and tLH-R trunc ) could be particularly important as receptor variants lacking the TM and IC region, since the secreted proteins would be capable of binding LH [34].…”

“…For example, a marked increase in the circulating levels of PRL has been associated with incubation behavior, cessation of egg laying, and ovarian regression in the domestic turkey [15]. Exogenous administration of mammalian PRL has been shown to induce incubation behavior and/or gonadal regression and to cause a decline in circulating estrogen due to suppression in steroidogenic enzyme gene expression [15][16][17][18][19]. Ovarian regression may be achieved by a direct effect of PRL or may be attributable to reduced numbers of ovarian LH-Rs or both [18,19].…”

“…Exogenous administration of mammalian PRL has been shown to induce incubation behavior and/or gonadal regression and to cause a decline in circulating estrogen due to suppression in steroidogenic enzyme gene expression [15][16][17][18][19]. Ovarian regression may be achieved by a direct effect of PRL or may be attributable to reduced numbers of ovarian LH-Rs or both [18,19]. The biological actions of gonadotropins within the hen ovary are associated with gonadotropin-induced steroid synthesis and corresponding steroidogenic gene expression [20].…”

Expression and Localization of Prolactin Receptor Messenger Ribonucleic Acid in Red Deer Ovary During the Estrous Cycle and Pregnancy1

“…Suppression of this elevation in PRL secretion by bromocriptine administration has been shown to delay significantly the onset of anestrus in red deer hinds [3], suggesting that the breeding season is terminated partly by the effect of seasonal hyperprolactinemia. PRL may exert this effect directly at the level of the ovary through modulation of LH receptor (LH-R) expression [4]. Alternatively, elevated secretion of PRL may affect the GnRH axis through interaction with PRL receptors (PRL-R) in the brain [5].…”