POLYPLOIDY IN THE FRESHWATER SNAIL LYMNAEA STAGNALIS (GASTROPODA, PULMONATA). A CYTOPHOTOMETRIC ANALYSIS OF THE DNA IN NEURONS AND SOME OTHER CELL TYPES

by

H. H. BOER, C. GROOT, M. DEJONG-BRINK and C.J. CORNELISSE (Biological Laboratory,Free University,Amsterdam and Departmentof Pathology, Universityof Leiden, The Netherlands)

SUMMARY

Feulgen-stained nuclei of neurons and of some other cell types of the freshwater snail Lymnaeastagnalis were measured cytophotometrically. The DNA content of neurons doubles a number of times during the life of the snail. Randomly chosen neuronals nuclei appeared to fall within distinct DNA classes. In 30 mm (shell height) animals all classes up to that of 4096C are present in the central ganglia. It was estimated that a 4096C nucleus contains 2.4 × 10-3 µg DNA. The functional significance of neuronal polyploidy is not clear. Intestinal epithelium cells are diploid. Intestinal gland cells, Sertoli cells, pore cells and Calcium cells attain DNA values of 16C.

INTRODUCTION

In the central nervous system of opisthobranch and pulmonate gastropods giant neurons occur in addition to small and middle- sized cells (BULLOCK & HORRIDGE, 1965; SANCHIS & CASTRO, 1972). Probably most of the neurons are polyploid, albeit to different degrees. Indirect substantiation of this hypothesis has been presented by BOER (1965), who concluded on the basis of morphometrical data that in the pond snail Lymnaea stagnalis ordinary as well as neurosecretory neurons grow by duplicating their cell- and nucelar volumes a number of times in the course of life. A more direct approach to the problem is the performance of DNA measurements. Such studies have been carried out on the neurons of a number of gastropod species (KUHL- MANN, 1969; COWDEN, 1972), in particular on those ofAplysia californica (COGGESHALL et al., 1970; LASEK, 1971; LASEK & DOWER, 1971). It has been established that the DNA content of an identified neuron of this opisthobranch (R-2 of the abdominal ganglion) increases step-wise as the animal grows; obviously at each step the whole genome is duplicated (COGGESHALL et al., 1970). R-2 and other giant cells containing amounts of 131 X 10-3 DNA per cell are not rare in large (600 gm) animals; it could be deduced that in these nuclei the 246

2C DNA had duplicated as many as 16 times. In addition to these giant nuclei cells with lower degrees of polyploidy were recognized (LASEK, 1971; LASEK & DOWER, 1971). In the present study DNA measurements were carried out on neurons of L. stagnalis ; this species is extensively studied both morphologically and physiologically (e.g. BOER & JoossE, 1975). In addition to neurons some other cell types were investigated.

MATERIALS AND METHODS

The snails used for the investigations were taken from stock bred in the laboratory. Sizes of snails given below refer to shell height. Small pieces of tissue (volume < 1 mm3) treated with trypsin (0.25 % in snail Ringer) for 1 hr at 37° C, were squashed on soft edge glass slides (Socorex) in a drop of snail Ringer by pressing them gently with a cover slip. The preparations were then frozen with Cryokwik (IEC Catalog no. 3377, Needham HTS, Mass., U.S.A.) and the cover slips were removed with a razor blade. Fixation was carried out for 1 hr at room temperature in Carnoy's fixative. To stain DNA a slightly modified Feulgen procedure was used (DUIJNDAM & VAN DUIJN, 1973). Hydrolysis in 5N HCl and staining with the Schiff reagent were both performed at room temperature for 1 hr. After rinsing in sulphite solution and dehydration, the prepara- tions were mounted in Fluoromount or in Caedax (ND 1.55). Scanning microdensitometric measurements of the Feulgen-stained cell nuclei were performed with a Leitz-MPV II microphotometer equipped with a 0.5 ym Zeiss scanning stage, interfaced to a PDP 11/10 computer (Digital Equipment Corporation, Maynard, Mass., U.S.A.). A pseudogrey value image of the scanned array was displayed on a Tektronix 4010 graphic terminal. Measurements were carried out with light of 558 nm, which was selected from a quartz halogen lamp (Philips 12V, 100W, 7023) with an interference filter AL 558 nm (Schott, Mainz, Germany). Measurements of the integrated absorp- tion of the Feulgen-stained nuclei were performed with the Histo- chemical Data Acquisition Program (VAN DEN BROEK, 1976). Cells were scanned with a 0.5 fLm step size and 0.6 ym spot size. For very large nuclei of neurons the step size had to be increased to prevent overflow of the computer memory which had a limited capacity of 60 X 60 picture points only. In that case the integrated absorption was multiplied by the square of twice the step size in ym. The HIDACSYS program contains an image editing routine for erasing dirt and touching nuclei of other cells from the digitized image of the scanned nucleus.