Morphogenesis of the residual body of the mouse testis

Transcription

1 93 Morphogenesis of the residual body of the mouse testis By CASIMIR F. FIRLIT and JOSEPH R. DAVIS (From the Department of Pharmacology and Therapeutics, Stritch School of Medicine, and Graduate School, Loyola University, Chicago, U.S.A.) Summary A histochemical study of the detached cytoplasm of the maturing spermatid has been carried out. The detached cytoplasm has been designated as the cytoplasmic body and 4 distinct phases in its morphogenesis have been reported. The cytoplasmic tag of stage 11 of spermiogenesis has been designated as phase CB-i in the morphogenesis of the cytoplasmic body. It was found to contain numerous granules of RNA, polysaccharides, and lipid. During phase CB-2 and CB-3, coalescence and fusion of these granules occurs, which result in a centrally-oriented plaque of RNA and glycogen, peripheral satellites containing polysaccharides, and large globules of lipid. During phase CB-4, the cytoplasmic body membrane becomes approximated to the plaque and at this stage of development the cytoplasmic body can be compared to the 'residual bodies' of Regaud. Peripheral migration towards the basement membrane of the seminiferous tubule now occurs. Some of the residual bodies undergo phagocytosis by Sertoli cells, while others eventually occupy a position at the level of the spermatogonia. Introduction I T has long been known that during spermiogenesis, a considerable amount of cytoplasm is lost from the maturing spermatid. This detached cytoplasm was originally designated as the 'residual body' by Regaud (1901) and was subsequently shown by Daoust and Clermont (1955) to contain large aggregates of RNA in the form of separate granules. Recently, Lacy (1962) has demonstrated that the residual bodies found in adult rat testes contain significant amounts of lipid in addition to RNA. Although the process of spermatogenesis occurs in a cyclic manner in non-seasonal animals, the regulatory mechanisms have remained obscure. Roosen- Runge (195 2) has speculated that the zone of residual bodies may be associated with the control of a wellregulated pattern of spermatogenesis inasmuch as the residual bodies appear at the time of the elimination of the spermatozoa into the lumen of the seminiferous tubule. The present studies were designed to investigate the morphogenesis of the residual body of the mouse testis. It was found that the development of the residual body during spermiogenesis could be divided into 4 major phases, each of which displayed a characteristic histochemical staining pattern. Methods Male Swiss mice, approximately 20 weeks in age, were killed by decapitation. The testes were immediately removed and placed in cold Carnoy's solution and left overnight. The fixed tissue was dehydrated in 3 changes of [Quart. J. micr. Sci., Vol. 106, pt. 1, pp , 1965.]

2 94 Firlit and Davis Residual body of mouse testis cold absolute ethanol, cleared in methyl salicylate, and embedded in 'tissuemat'. Tissues were sectioned at 8 \i and mounted on glass slides. The Feulgen technique (Stowell, 1945) was used to show the localization of DNA. As a control, unhydrolysed sections were placed in distilled water at 6o C for 8 min before staining with Schiff's reagent. The methyl green / pyronin Y method as modified by Taft (1951) was employed to demonstrate the presence of both DNA and RNA. Feulgen's technique confirmed the localization of DNA as indicated by methyl green, and digestion in protease-free ribonuclease confirmed the localization of RNA as indicated by pyronin Y. The periodic acid/schiff reaction (McManus, 1948) was employed to demonstrate polysaccharides. Best's (1903) carmine was used to confirm the presence of glycogen as demonstrated by the PAS reaction. As a control procedure for Best's carmine a 20-min digestion at 37 0 C was carried out with a 0-5% solution of saline-activated malt diastaste. In order to localize RNA and PAS-reactive material simultaneously, a PAS / toluidine blue staining combination was employed. The localization of RNA by toluidine blue was found to be identical to that found with pyronin Y. For the demonstration of lipid material, the testis was fixed in cold 10% neutral formalin overnight and embedded in gelatin. The tissues were cut on a freezing microtome and visualization of the lipid material carried out by colouring with Sudan IV. Results The morphological classification of spermiogenesis in the mouse as proposed by Oakberg (1956) has been used in the present report. During the development of the spermatid, a considerable amount of cytoplasm is lost. The remnant of the cytoplasm detached from the spermatid has long been known as 'the residual body'. The data of the present study indicate, however, that the residual body is only the final stage in a complex series of morphological changes occurring in the discarded cytoplasm of the spermatid. Consequently, the cytoplasm released from the spermatid has been designated as the 'cytoplasmic body' (CB), and its morphogenesis has been divided into 4 major phases, which are summarized in fig. 1. Phase CB-i in the morphogenesis of the residual body. Fig. 2, A shows Oakberg's stage 10 of spermiogenesis in the mouse. Only a small amount of RNA-staining material (arrows) occurs in the cytoplasm of the spermatid during the development of the cytoplasmic tag. However, in the further course of spermiogenesis, this RNA-positive material appears to coalesce into separate granules as illustrated in fig. 2, B. This coalescence occurs at stage 11 of spermiogenesis which has also been designated in the present report as phase CB-i in the morphogenesis of the residual body of the mouse testis. In addition, the entire cytoplasmic tag in phase CB-i stains with the PAS reaction. Two distinct types of granulations were observed with this stain: (1) numerous very small PAS-positive granules scattered uniformly throughout the cytoplasm of the spermatid, and, (2) larger, lightly staining PASpositive granules, also found in the cytoplasm of the spermatid. When

3 Firlit and Davis Residual body of mouse testis 95 sections were stained with Best's carmine and digested with malt diastase, only the larger PAS-reactive granules were found to contain glycogen. The RNA-g KNA-p _... A4S-S > '" ~PAS-g t-^-' PAS-p W^-RNA-p g'/-s gly-p HNA-g & «{ FIG. 1. Diagram of the 4 phases in the morphogenesis of the residual body, cb, cytoplasmic body; ct, cytoplasmic tag; gly-g, glycogen granule; gly-p, glycogen plaque; Id, lipid droplet; Ig, lipid globule; m, membrane of cytoplasmic body; PAS-g, small PAS-positive granule; PAS-G, large PAS-positive granule; PAS-p, PAS-positive plaque; PAS-s, PAS-positive satellite; rb, residual body; RNA-g, RNA-positive granule; RNA-p, RNA-positive plaque; scm, spermatid cell membrane; sn, spermatid nucleus; sp, cytoplasmic body space. RNA-positive granules increase in size during late phase CB-i in the morphogenesis of the residual body. Frozen sections coloured with Sudan IV demonstrated finely dispersed lipid droplets throughout the spermatid cytoplasm. Phase CB-2 in the morphogenesis of the residual body. When the cytoplasmic tag (phase CB-i) becomes detached from the maturing spermatid, this structure has been designated as the cytoplasmic body in the present report.

4 o.6 Firlit and Davis Residual body of mouse testis Fig. 2, D shows the cytoplasmic body (CB), which is now phase CB-2 in the morphogenesis of the residual body. The remnant of the cytoplasmic membrane of the maturing sperm becomes the membrane of the cytoplasmic body; it is PAS-positive. The RNA-positive granules continue to coalesce and will ultimately form the RNA-plaque at phase CB-3. On the other hand, the smaller PAS-positive granules continue to coalesce into larger granules which become located along the periphery of the cytoplasmic body in phase CB-3 an^ ultimately form the PAS-satellites. The larger PAS-positive granules that stained positively with Best's carmine are also found ultimately to fuse to form part of the plaque area visible in phase CB-3. ^n addition, the lipid droplets are observed to coalesce further into larger globules located in a random fashion throughout the cytoplasmic body. By late phase CB-2, these lipid globules have migrated towards the cytoplasmic body membrane. Phase CB-3 * n the morphogenesis of the residual body. Fig. 2, E shows early phase CB-3 m tne morphogenesis of the residual body. The coalescence of the RNA-positive granules has continued until at this point of development a single large RNA-plaque (/>) is visible within the cytoplasmic body. The coalescence of the smaller PAS-reactive granules during their peripheral migration has also resulted in the formation of larger PAS-satellites (s) which are now in apparent contact with the membranes (m) of the cytoplasmic body. Fig. 2, F illustrates late phase CB-3 in the morphogenesis of the residual body. The plaque area of the cytoplasmic body at this period in development stains very intensely with pyronin Y as well as with PAS. In addition, the RNA-plaque now shows numerous indentations. Sections coloured with Sudan IV / toluidine blue at this point of development (fig. 3, A, B) reveal the presence of lipid-positive material (arrow) in the cytoplasmic body space. These lipid globules appear to be responsible for the indentations of the plaque area. Fig. 3, c shows that these large lipid globules are found within the cytoplasmic bodies that are located along the periphery of the lumen of FIG. 2 (plate). A, stage 10 (Oakberg, 1956) of spermiogenesis in the mouse. Note lack of granulations in the cytoplasmic tags (arrows). Toluidine blue. B, stage 11 of spermiogenesis in the mouse (early phase CB-i in the morphogenesis of the cytoplasmic body). Note the appearance of RNA granules (arrows) in the cytoplasmic tag (ct). PAS / toluidine blue. C, stage 14 of spermiogenesis in the mouse (late phase CB-i in the morphogenesis of the cytoplasmic body). Note the decrease in number of the RNA granules (arrow) in the cytoplasmic tag (ct). Toluidine blue. D, stage 16 of spermiogenesis in the mouse (phase CB-2 in the morphogenesis of the cytoplasmic body). The detached cytoplasm of the maturing spermatids has been designated as the cytoplasmic body (cb) in the present report. Note the increase in size along with a decrease in the number of the RNA granules. Toluidine blue. E, early phase CB-3 in the morphogenesis of the cytoplasmic body. Note the single large RNA plaque (p) in the cytoplasmic body and the PAS satellites (s). PAS / toluidine blue. F, late-phase CB-3 in the morphogenesis of the cytoplasmic body. The RNA plaque (p) now shows indentations. Note the numerous PAS satellites (s) which have also increased in size and lie close to the membrane (m) of the cytoplasmic body. PAS / toluidine blue.

5 FIG. 2 C. I". FIRLIT and J. R. DAVIS

6 Kio. 3 C. F. F1RLIT and J. K. DAVIS

7 Firlit and Davis Residual body of mouse testis 97 the seminiferous tubule. From this position, migration of the cytoplasmic bodies begins towards the basement membrane of the tubule. At the onset of this migration, the lipid material begins to decrease, and simultaneously with this decrease the membrane of the cytoplasmic body collapses, resulting in a decrease in the cytoplasmic body space as well as a condensation of the PAS-satellites on the now spherical RNA-plaque at the initiation of phase CB-4. At this point in its development, the cytoplasmic body takes on the appearance of the classical 'residual body' and has been so designated in the present report. Phase CB-4 i n the morphogenesis of the residual body. Two fates of the residual body can be demonstrated during its migration from an initial position along the periphery of the lumen of the tubule at phase CB-3 to its final position near the basement membrane during phase CB-4. The first fate of the residual body involves its phagocytosis by a Sertoli cell as illustrated in fig. 3, D, E. Further changes in the structure of the cytoplasmic body subsequently occur within the Sertoli cytoplasm. The cytoplasmic body membrane becomes approximated to the plaque, resulting in the obliteration of the cytoplasmic body space. This obliteration would appear to be due to the removal of the lipid globules from the cytoplasmic body space by the Sertoli cell, resulting in the accumulation of finely-dispersed lipid granules in the basal regions of the Sertoli cytoplasm. The second fate of the residual body involves its migration towards the basement membrane of the seminiferous tubule without undergoing phagocytosis by a Sertoli cell (fig. 3, F). During this migration of the residual body, similar morphological changes occur as previously described, including the loss of lipid granulations in the regions of the spermatogonia and primary spermatocytes. After these morphological changes, the residual bodies continue to migrate towards the level of the spermatogonia (fig. 3, G) and finally to occupy a position against the basement membrane of the seminiferous tubule (fig. 3, H). FIG. 3 (plate), A, late phase CB-3 ' n the morphogenesis of the cytoplasmic body, coloured with Sudan IV / toluidine blue. The arrow indicates sudanophilia in the cytoplasmic body space. Note that the lipid material produces the indentations of the RNA plaque (p). B, late Phase CB-3 i n the morphogenesis of the cytoplasmic body, coloured with Sudan IV / toluidine blue, showing lipid material (arrows) in cytoplasmic bodies along the periphery of the lumen of the seminiferous tubule. C, photomicrograph at lower magnification of the seminiferous tubule, stained with pyronin Y. Note the localization of the cytoplasmic bodies round the lumen during phase CB-3. D, phase CB-4 m the morphogenesis of the cytoplasmic body. When the membrane of the cytoplasmic body is no longer visible, the cytoplasmic body has been designated as the residual body (rb) in the present report. Note the Sertoli cell phagocytosing a residual body. Iron haemotoxylin. E, same section as D at higher focus. The Sertoli cytoplasm (c) passes over the residual body (rb). F, Decrease in the staining intensity of the RNA plaque with toluidine blue in phases CB-3 and CB-4 in the morphogenesis of the cytoplasmic body. o, migration of residual bodies (arrows) to the level of the spermatogonia. Iron haematoxylin. H, migration of residual bodies (arrows) to the level of the basement membrane. Iron haematoxylin H

8 98 Firlit and Davis Residual body of mouse testis Discussion Numerous attempts have been made to formulate a 'trigger' mechanism which would regulate the periodicity of the spermatogenic cycle. Roosen- Runge (1952) has speculated that the release of older spermatids from the lumen of the seminiferous tubule is in some unknown manner associated with the regulation of the development of earlier generations of the germinal epithelium. Lacy (1962) demonstrated that the bulk of the lipid observed in the Sertoli cells was derived from the phagocytosis of the residual bodies, and that after the release of the spermatozoa, there was a gradual reduction in the lipid content of the Sertoli cell, indicating the release of some substance that might influence spermatogenesis. For these reasons, it was postulated that the local regulating mechanism involved in the successive replacement of germ cells about a radial axis during the spermatogenic cycle may be due to a Sertoli cell hormone, the production of which is initiated or accelerated by the phagocytosis of the residual bodies. The data of the present studies indicate that glycogen is a significant constituent of the cytoplasmic body. After the development of the cytoplasmic body into the residual body at phase CB-4, two possible fates of this structure have been demonstrated. The residual body can either be phagocytosed by a Sertoli cell or can independently migrate to a final position along the basement membrane of the seminiferous tubule. In either case the possibility exists that the residual body may contribute appreciable amounts of glycogen either to the Sertoli cell or to the germinal epithelium (Nicander, *957)- One of us (C. F. F.) was supported by a Lederle Medical Student Research Fellowship. This work was supported by an Institutional Grant from the General Research Support Grant of the Public Health Service and The Anna Fuller Fund. The authors wish to express their appreciation to Dr. K. K. Hisaoka of the Department of Biological Sciences, Loyola University, for his many helpful suggestions. References BEST, A., Beitr. path. Anat. allg. Pathol., 33, 585. DAOUST, R., and CLERMONT, Y., Amer. J. Anat., 96, 255. LACY, D., Brit. med. Bull., 18, 205. MCMANUS, J. F. A., Stain Tech., 23, 99. NICANDER, L,., Acta neerl. morph., 1, 233. OAKBEEC, E. F., Amer. J. Anat., 99, 391. REGAUD, C, Arch. d'anat. micr., 4, 101. ROOSEN-RUNCE, E. C, Ann. N.Y. Acad. Sci., 55, 574. STOWELL, R. E., Stain Tech., 20, 45. TAFT, E. B., Stain Tech., 26, 205.