SHORT NOTE Sudden Volume Changes of the Lumen of Inverted Thyroid Follicles in Suspension Cultures Use in Estimation of Rates of Water Flux Through Thyroid Epithelium LUCIO NITSCH’ and SEYMOUR H. WOLLMAN*** ‘Centro di Endocrinologia ed Oncologia Sperimentale de1 CNR, Dipartimento di Biologia e Patologia Cellulare e Moleculare, II Facoltrl di Medicina, 80131 Naples, Italy, and ‘Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Inverted thyroid follicles in suspension culture were observed using dark-field illumina- tion and time-lapse video enhancement techniques. The lumens of many follicles undergo episodes of rapid shrinkage followed by relatively slow dilation. The extent of shrinkage ranged from 10 to 40% of the initial diameter. The decrease in lumen diameter is accompanied by loss of luminal fluid. The rate of efflux of fluid amounted to from 1 to 90 &/pm’ of luminal surface/min. This was followed after several minutes by a slow and generally linear increase in lumen diameter with time that could be accounted for by an influx of fluid of the order of 0.5 pm’lpm*/min. It is possible that this phenomenon is due to a continued influx of fluid into the lumen until a junction opens between cells allowing a sudden loss of luminal fluid. The junction reseals and fluid begins to accumulate in the lumen. The phenomenon may be a three-dimensional analogue of the behavior of do- mes. Q 1986 Academic Press, Inc. When separated rat thyroid follicles [l] are cultured in suspension in a medium containing 5 % calf serum [2, 31, they invert (turn inside-out). In a suspension of such inverted follicles, most have large electron-lucent lumens D-51 surrounded by flat, attenuated cells [3]. Although there are some inverted follicles that are relatively small, with small lumens [6], there is evidence to suggest that the attenuated cells are stretched in the plane of the follicle wall by fluid that is secreted into the lumen [3, 51. We have now observed that these inverted follicles undergo episodes of relatively rapid shrinkage followed by slow dilation and have used these observa- tions to estimate the rate of influx of water into the lumen that occurs during the dilation. Materials and Methods Follicles were prepared from the thyroid gland of young male Fischer rats weighing 100-120 g as previously described [I]. The follicles were cultured in suspension in Coon’s modified Ham F12 medium containing 5% calf serum. After 3 days, the medium was changed. Two days later the inverted follicles were suspended in a 35-mm plastic Petri dish (Costar) coated with agarose to prevent adhesion to the plate. They were observed using darkfield illumination. Recordings were made using time-lapse video-enhanced image intensification [7, 81 taking one frame every 8 sec. During record- ings the follicles in the dish were in a chamber at 37°C and were exposed to an atmosphere of air containing 10% CO*. We are indebted to Dr Mark C. Willingham for making his apparatus available to us and for instructing us in its use. * To whom offprint requests should be sent. Address: National Institutes of Health, Bldg 37, Rm lE16, Bethesda, MD 20892, USA. Copyright @ 1986 by Academic Press, Inc. All ri!ghts of reproduction in any form reserved WJl4-4827/86 SO3.00