107

J. Anat. (1979), 128, 1, pp. 107-119 With 13 figures Printed in Great Britain

An ultrastructural study of the testis and epididymis of the rat after treatment with prostaglandins E2 and F2,. (PGE2 and PGF2,J E. C.-F. TSO* AND D. LACY

Department ofZoology and Comparative Anatomy, St Bartholomew's Hospital Medical College, Charterhouse Square, London E. C. 1

(Accepted 19 January 1978) INTRODUCTION

The injection of the prostaglandin PGE2 into the rat causes impairment of spermatogenesis (Ericsson, 1972; Tso, 1976) and the appearance of amorphous bodies (or droplets) and young germ cells in the epididymis (Tso, 1976). The present report is an account of the ultrastructural changes in the testis after injections of PGE2 and PGF2O1. An attempt has also been made to identify the amorphous bodies in the epididymis. MATERIALS AND METHODS

Adult Wistar male rats weighing 250-280 g were divided into five experimental groups, each consisting of at least five animals. A single dose of either PGE2 or PGF23, in 0 1 ml sterile saline was injected into the right testis of each experimental animal. The various dosages used were: Group I, 5 ,cg PGE2; Group II and V, 100 ,ug PGE2; Group III, 5,ug PGF2a; Group IV, 100 4ug PGF2,. The left testis served as an additional control for possible systemic effects. Animals in Groups I-IV were killed 4 hours, and those in Group V 24 hours, after the injection. Animals in the control groups were injected in the same way with 0 1 ml sterile saline only. After the animals had been killed, their testes and epididymides were dissected out and processed as follows: The testis was trimmed into 1 mm cubes, fixed in ice-cold Millonig's phosphatebuffered glutaraldehyde for 2-3 hours, rinsed in the same buffer (pH 7 3) for 1 hour, then post-fixed for 1 hour in Millonig's phosphate-buffered osmium tetroxide at 4 'C. The tissue was then dehydrated through a graded series of alcohols and finally infiltrated and embedded in Araldite. Thin sections showing silver-gold interference colour were prepared with an LKB ultratome using a glass knife and picked up with naked copper grids. The sections were stained with saturated uranyl acetate for 15-30 minutes and then in lead citrate for 30 minutes. These were then viewed under a Jeol Jem 7A electron microscope. The epididymis was processed as described by Flickinger (1972). Large pieces of the epididymis were fixed in ice-cold Karnovsky's (1965) fixative which contained 5 % glutaraldehyde and 4 % formaldehyde in 01 M cacodylate buffer (pH 7 3) for 1 hour. The tissue was then cut into 1 mm cubes and immersed in the same fixative for a further hour at 4 °C, then rinsed in distilled water and post-fixed in cold 1 % * Present address: Zoology Department, University of Western Ontario, London, Ontario N6A 5B7, Canada.

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osmium tetroxide in 0'1 M cacodylate buffer for 1 hour. The tissue was dehydrated and processed in the same manner as the testis. Thick sections of the testis and epididymis were cut and stained with 0-1 % aqueous toluidine blue solution for light microscopy. Stages of development within the seminiferous epithelium were identified according to the schedule of Leblond & Clermont (1952). RESULTS

In general, the ultrastructure of the boundary tissue in the testes treated with PGE2 and PGF2, did not differ from that seen in the contralateral testes or in the control animals, and resembled that described by previous workers (Lacy & Rotblat, 1960). However, in a number of sections from testes of different animals treated with PGF2c, (Groups III and IV), definite changes had occurred in the cells of the inner cellular layer (myoid cells). They displayed marked swellings along the outermost surface which protruded into the outer non-cellular layer of the boundary tissue (Fig. 1). The swellings contained an amorphous material of low electron density, and were apparently devoid of fine filaments. Organelles resembling multivesicular bodies were observed in the myoid cells of both experimental and control testes (Fig. 2). Such structures appear not to have been reported previously. They consisted of a group of small vesicles and some dense granules enclosed within a single membrane. Very often they were found closely associated with other cytoplasmic vesicles. Although only observed in a few sections of the testes treated with PGF2

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118

E. C.-F. TSO AND D. LACY

disruption of the seminiferous epithelium and exfoliation of part of the germinal epithelium. In rats, after the administration of prostaglandins E1, E2 and F2a through the testicular vein, testicular artery, or directly into the intertubular tissue, it has been reported that the testicular venous pressure is increased, while there is a reduction in blood flow and arterial side pressure (Free & Jaffe, 1972). In view of this, the prostaglandins injected in the present study have probably caused a disturbance in the vascular system of the testis (as suggested by Ericsson, 1972 and Memon, 1973), followed by the effects aforementioned. It is also worth mentioning that most of the major events observed here, such as the changes in the myoid cells, the damage to germ cells, the formation of multinucleate spermatids, and the exfoliation of part of the germinal epithelium, are similar to those reported after the ligation of the testicular artery (Kaya & Harrison, 1975). This is further evidence in favour of the explanation of our results given above. It is interesting that the spermatocytes were particularly susceptible to damage by prostaglandins PGE2 and PGF2a. It has been reported that an electron-dense junction occurs between the Sertoli cell and spermatogonia and spermatocytes (Brokelmann, 1963; Nicander, 1967; Fawcett, 1975; Kaya & Harrison, 1976; Russel, 1977), and, as the germ cells develop into elongated spermatids, a specialized junction is formed on the Sertoli cell side of the junction (Nicander, 1967; Fawcett, 1975). According to Ross & Dobler (1975), this specialized junction begins to develop as early as the spermatocyte stage. This has also been confirmed by one of the present authors (Tso, 1975). Though postulations regarding the function of the junction have been made (Flickinger & Fawcett, 1967; Russel, 1977), its exact significance remains obscure. Nevertheless, it seems reasonable to suggest that the change-over of the electron-dense junction to the specialized junction may represent an alteration in the physiological or anatomical relationship between the Sertoli cell and the developing germ cell, or between the germ cell and the external environment in general, and this may possibly be related to the special susceptibility of the spermatocytes to exogenous prostaglandin. During the course of this study certain organelles, such as membrane-bound granular bodies and multivesicular body-like structures, were observed in the myoid cells, and these deserve attention. Membrane-bound granular bodies in the pancreas and prostate are believed to be connected with the process of secretion (Swift & Hruban, 1964), while multivesicular bodies have been equated with digestive vacuoles and secondary lysosomes (Novikoff, Essner & Quintana, 1964). The presence of similar organelles in the myoid cells clearly requires further study. SUMMARY

Intratesticular injections of PGE2 and PGF2, caused damage to germ cells and exfoliation of part of the damaged germinal epithelium into the epididymis. Membrane-bound granular bodies and localized swellings were also seen in some of the myoid cells in experimental animals. However, the Sertoli cells and Leydig cells remained unaffected. We are grateful to Dr J. E. Pike for the generous gift of PGF2a, and PGE2. The work was carried out during the tenure of a Commonwealth Scholarship by E. C.-F. T.

Prostaglandins on testis

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RE FERENCES

BROKELMANN, J. (1963). Fine structure of germ cells and Sertoli cells during the cycle of the seminiferous epithelium in the rat. Zeitschrift fur Zellforschung und mikroskopische Anatomie 59, 820-850. ERICSSON, R. J. (1972). Prostaglandins (E1 & E2) and reproduction in the male rat. Advances in Biosciences 9, 737-742. FAWCETT, D. W. (1975). The ultrastructure and functions of the Sertoli cell. Handbook ofPhysiology, vol. 5, sect. 7, pp. 21-55. Washington, D.C.: Physiological Society. FLICKINGER, C. J. (1972). Alterations in the fine structure of the rat epididymis after vasectomy. Anatomical Record 173, 277-300. FLICKINGER, C. J. & FAWCETT, D. W. (1967). The junctional specializations of Sertoli cells in the seminiferous epithelium. Anatomical Record 158, 207-222. FREE, M. J. & JAFFE, R. A. (1972). Effect of prostaglandins on blood flow and pressure in the conscious rat. Prostaglandins 1, 483-498. KARNOVSKY, M. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolarity for use in EM. Journal of Cell Biology 27, 137A. KAYA, M. & HARRISON, R. G. (1975). An analysis of the effect of ischaemia on testicular ultrastructure. Journal of Pathology 117, 105-117. KAYA, M. & HARRISON, R. G. (1976). The ultrastructural relationships between Sertoli cells and spermatogenic cells in the rat. Journal of Anatomy 121, 279-290. LACY, D. & ROTBLAT, J. (1960). Study of normal and irradiated boundary tissue of the seminiferous tubules of the rat. Experimental Cell Research 21, 49-70. LEBLOND, C. D. & CLERMONT, Y. (1952). Definition of the stages of the cycle of the seminiferous epithelium in the rat. Annals of the New York Academy of Sciences 55, 548-573. MEMON, G. N. (1973). Effects of intratesticular injections of prostaglandins on the testes and accessory sex glands of rats. Contraception 8, 361-370. NICANDER, L. (1967). An electron microscopical study of cell contact in the seminiferous tubules of some mammals. Zeitschriftftir Zellforschung und mikroskopische Anatomie 83, 375-397. NOVIKOFF, A. B., ESSNER, E. & QUINTANA, N. (1966). Golgi apparatus and lysosomes. Federation Proceedings 23, 1010-1022. NYQUIST, S. E., AcLW, K. & MOLLENHAUER, H. H. (1973). Residual bodies and their components. I. Isolation methods. Biology of Reproduction 8, 119-124. Ross, M. H. & DOBLER, J. (1975). The Sertoli cell junctional specializations and their relationship to the germinal epithelium as observed after efferent ductule ligation. Anatomical Record 183, 267-292. RUSSEL, L. (1977). Movement of spermatocytes from the basal to the adluminal compartment of the rat testis. American Journal of Anatomy 148, 313-328. SwIFr, H. & HRUBAN, Z. (1964). Local degradation as a biological process. Federation Proceedings 23, 1026-1037. Tso, E. C.-F. (1975). The effects of prostaglandins on the male reproductive system of the rat. Ph.D. thesis, University of London. Tso, E. C.-F. (1976). Effects of prostaglandin E2 (PGE2) on the male reproductive system. Contraception 14, 53-59. Tso, E. C.-F. & LACY, D. (1975). Effects of prostaglandin F2,, on the reproductive system of the male rat. Journal of Reproduction and Fertility 44, 545-550.

An ultrastructural study of the testis and epididymis of the rat after treatment with prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha).

107 J. Anat. (1979), 128, 1, pp. 107-119 With 13 figures Printed in Great Britain An ultrastructural study of the testis and epididymis of the rat a...
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