Separation and maturation of gonadotrophs from 2A8 clonal cells in vitro

Summary 2A8 clonal cells derived from the epithelium of Rathke's pouch of fetal rats were cultured in growth medium supplemented with fresh rat serum, median-eminence extract and l-thyroxine. Then, in order to isolate gonadotrophs, cyanogen bromide-activated Sepharose which was conjugated with LHRH (LHRH-Sepharose) was added to the culture medium. Fourteen days after incubation of 2A8 cells with LHRH-Sepharose, agranular and granular cells were rapidly bound to LHRH-Sepharose when fresh serum had previously been added in the medium. The cytoplasm of granular cells that were bound to LHRH-Sepharose contained spherical secretory granules (200–250 nm in diameter). These cells were similar in morphology to the FSH and LH gonadotrophs described by Kurosumi (1968). Moreover, many of them appeared as hypertrophied cells that resembled castration cells. These results demonstrate that LHRH conjugated to Sepharose can be used to separate gonadotrophs from other 2A8 cells, and it is suggested that the hypertrophy of some cells might be due to persistent stimulation by LHRH which is likely bound to receptors on the cell membrane.Supported by an Institutional Research Grant from The University of Texas Health Science Center at San Antonio, San Antonio, TexasThe authors wish to thank Mrs. Pauline Polette for her skillful technical assistance. Human median eminence extract was kindly supplied by Dr. A. Takatsu, Department of Regal Medicine, Tokyo University Faculty of Medicine, Tokyo, Japan     

pH-dependent inhibition of mushroom tyrosinase by N-substituted N-nitrosohydroxylamines

Several synthetic N-substituted N-nitrosohydroxylamines were found to inhibit mushroom tyrosinase in a pH-dependent manner regardless of the N-substituent. The inhibitory activity, or pI(50) ( - log [IC(50), M]) value, linearly decreased as the pH of the media increased. The inhibitory activities of tested N-substituted N-nitrosohydroxylamines at pH 6.8 and 5.8 were found to be almost 10 times and 100 times greater than at pH 7.8, respectively. The types of inhibition were different at pH 6.8 and 5.8. These results suggest that the inhibitory effect of N-substituted N-nitrosohydroxylamines is caused by the non-ionized form of the inhibitor. Furthermore, the mechanism of inhibition depends on the interaction between the inhibitor and the active site of tyrosinase at different pH values.     

Gonadotroph-rich cell lines derived from pituitary clonal cells (2A8) grafted under the kidney capsule

Summary Gonadotroph-rich cell lines were established from multipotential pituitary clonal cells (2A8) which were implanted under kidney capsule of hypophysectomized female rats. These cell lines secrete gonadotrophins (FSH and LH) continuously over two months after establishment; LHRH stimulated the secretion of hormones into the culture medium. Many of the cells reacted immunohistochemically to antiserum to FSH or LH, while a small number reacted to antiserum to prolactin or TSH. They did not contain normal secretory granules such as those of gonadotrophs in vivo.Supported by USPHS Grant HD 11826 and NIH Grant P30 HD 10202. The authors wish to thank James Chambers (Immunocytochemistry), and Pat Koym and John Rhode (Radioimmunoassay) for their excellent technical assistance. We also express our thanks to NIAMDD for providing pituitary hormones     

Microtubules in thyroidectomy cells of the rat anterior pituitary gland.

Microtubules were successfully illustrated in thyrotrophs and thyroidectomy cells of rat pituitary glands. In contrast, microfilaments were mostly seen in the nonglandular follicular cells. Numerous microtubules were observed in the early stages of development of the thyroidectomy cells. In thyroidectomy cells microtubules were located in close proximity to mitochondria, endoplasmic reticula, secretory granules, and membranes of Golgi complexes. Consequently, it is suggested that microtubules may play a role in degranulation or other processes associated with the hypersecretory state.     

Electron microscopic immunocytochemistry of prolactin secreting cells (PRL-cells) and growth hormone secreting cells (GH-cells) in the anterior pituitary gland of partially hepatectomized rats

After 60% hepatectomy in rats, prolactin secreting cells (PRL-cells) and growth hormone secreting cells (GH-cells) of the anterior pituitary gland were distinctly identified by the protein A-gold procedure combined with electron microscopy. The animals were sacrificed by the decapitation at midnight at intervals of about 28, 76 and 124 h after the operation. The principal changes can be summarized as follows; (1) Hypertrophy of the Golgi complex, (2) Dilation of the rough endoplasmic reticulum (RER), (3) Increased numbers of secretory granules (markedly in GH-cells), and occurrence of granule extrusion or exocytosis, (4) Increased numbers of lysosomes, which were mostly seen at 124 h after the operation. These ultrastructural changes were remarkably observed in both PRL and GH-cells. Especially, the noticeable changes in PRL-cells after partial hepatectomy in the rat were new findings in this study. The above results suggest that hepatectomy induced synthesis and release of GH and PRL in cells from pars distalis.