Live Staining and Isolation of Specific Hormone-Producing Cells from Rat Anterior Pituitary by Cytochemistry with Lectins and Cholera Toxin B Subunit

Abstract

Anterior pituitary glands contain five types of hormone-producing cells. Distinguishing and isolating specific types of living cells are essential for studying their function. Although many such attempts have been made, the results have been disappointing. In the present study, we labeled specific types of living hormone-producing cells by using potential differences in sugar chains on the cell surfaces. Cytochemical analysis with lectins and cholera toxin B subunit revealed that PNA, S-WGA, and cholera toxin B subunit recognized sugar chains specific to prolactin cells, ACTH cells, and GH cells, respectively, and that UEA-I recognized most of prolactin cells and GH cells. Next, fluorescence-activated cell sorting was used to isolate GH cells labeled by fluoresceinated cholera toxin B. The purity of the GH cell fraction estimated by immunocytochemistry and quantitative real-time PCR for cell type-specific genes was more than 98%, which was higher than that reported in earlier studies, including those using transgenic animals. We conclude that cytochemistry with lectins and cholera toxin B subunit is a straightforward, acceptable method of isolating specific types of anterior pituitary cells and that the cells isolated by this method can serve as useful materials in the study of anterior pituitary cells.

Reassembly of Anterior Pituitary Organization by Hanging Drop Three-Dimensional Cell Culture

“…The cells were then resuspended in M199 (Life Technologies) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 100 units/ml penicillin, and 100 µg/ml of streptomycin (Life Technologies). Until this point, the protocol was identical to that for 2D cell culture [11]. For 3D culture, a 25 µl-drop containing 4000 cells was placed on the undersurface of plastic Petri dish lids (90 mm; Asahi Glass, Tokyo, Japan), which were then cultured over the sterile PBS (hanging drop) for 5 days at 37°C in a humidified incubator with 5% CO 2 (Fig.…”

<i>In Situ</i> Hybridization Method Reveals (Pro)renin Receptor Expressing Cells in the Pituitary Gland of Rats: Correlation with Anterior Pituitary Hormones

“…Sections were incubated overnight at room temperature in PBS with primary antibodies. Primary antibodies against the following proteins were used for immunostaining: adrenocorticotrophic hormone (ACTH), growth hormone (GH), prolactin, thyroid stimulating hormone (TSH) β-subunit, luteinizing hormone (LH) β-subunit as reported earlier [5, 12]. The antibody against LH β-subunit was used to identify the gonadotropes (LH/FSH cells).…”