An in vitro model for the effects of estrogen on neurons employing estrogen receptor-transfected PC12 cells

Abstract

Estrogen alters neurite outgrowth, neuritic spine development, and synaptogenesis in estrogen-responsive areas of the rat brain. However, examination of the specific effects of estrogen on neurons in vivo has been difficult. An in vitro model for the effects of estrogen on neurons was developed, using the PC12 rat pheochromocytoma cell line. Wild-type cells (PC12-WT) were stably transfected either with an expression vector coding for the full-length cDNA for the human estrogen receptor (hER), or with a control vector. Resultant clones were isolated, screened for incorporation of vector and expression of ER mRNA and protein, and analyzed for morphologic responses to estrogen. PC12-WT, NEO9 (ER-negative), and SER8 (ER-positive) cells exposed to 100 ng/ml NGF exhibited dose-responsive neurite outgrowth within 2 d by light microscopy (LM). Coadministration of 10(-10) to 10(-9) M estradiol (E2) had minimal effects on neurite outgrowth, neuritic spine development, or interneuritic connections in NEO9 or PC12-WT cells, but in SER8 cells E2 led to additive and dose-dependent increases in neurite outgrowth, spine development, and interneuritic connectivity. Coincubation of SER8 cells with E2 and the antiestrogen ICI 164,384 negated estrogenic effects on spine development and interneuritic connectivity. At the electron microscopic (EM) level, intercellular abutments of NEO9 or PC12-WT cells contained few and rudimentary gap junctions, with no increase by E2. However, SER8 cells exhibited augmented basal frequencies of gap junctions that increased with E2 incubation. Microinjection of Lucifer yellow into PC12-WT and NEO9 cells demonstrated low frequencies of dye coupling and no change with E2, but SER8 cells demonstrated increased dye-coupling frequency with E2 coincubation. The results suggest that SER8 cells recapitulate estrogen effects on neurons in vivo. Estrogen appears to induce an inherent neural morphologic program in estrogen receptor (ER)-containing cells. These three cell lines provide a unique in vitro system for studying mechanisms of estrogen-neuron interactions.

Environmentally relevant exposure to 17α-ethinylestradiol affects the telencephalic proteome of male fathead minnows

“…Estrogens have long been identified as hormones that promote neurite growth in vitro (Ferreira and Caceres, 1991; Lustig et al, 1994), a result consistent with the pathway analysis that EE 2 regulates cell differentiation and proliferation in the FHM telencephalon. Of specific interest, some proteins regulated by EE 2 were also involved in the processes of neuron network morphology and LTP.…”

Morphological castes in a vertebrate

“…Indeed, progesterone stimulates bone formation through the activation of gene expression for various growth factors, most notably insulin-like growth factors (24,25). Estrogen plays an important role in facilitating neuritic outgrowth and spinal chord formation (26) and thus also may be implicated in the elongation of the vertebrae and the spinal chord.…”

Dehydroepiandrosterone: A potential signalling molecule for neocortical organization during development

“…Experiments were performed at least three times for each treatment. To avoid counting bias and to ensure cells were not counted twice, we used the following parameters described (37): neurons whose somata abutted other somata were considered crowded and hence might not extend neurites optimally, and were not counted; cells that remained round and did not flatten on plastic were not counted; all other cells in the field were counted. A maximum of five cells per high-power field were counted; the field was moved in a discernible pattern to prevent recounting the same cells and to ensure that cells were sampled equally from the center and edge of the cultures.…”