Endoplasmic Reticulum-Golgi Apparatus Relationships in the Rat Spermatid L. HERMO, Y. CLERMONT AND A. RAMBOURG ' Department of Anatomy, McGill Uniuersity, Montreal, Quebec, Canada H3A 2B2
ABSTRACT The appearance of the spermatid Golgi apparatus was studied, both in thin sections of rat testes fixed in glutaraldehyde and treated with either tannic acid or ferrocyanide-reduced osmium, and in relatively thick sections (0.25-0.5pm) of glutaraldehyde fixed tissue impregnated with uranyl acetate followed by lead and copper citrate. With any one of these three procedures, the Golgi apparatus of young spermatids examined a t the Golgi and cap phases appeared as a compact hemispherical mass whose base was located next to the developing acrosomic system. The cortical region of the hemisphere was composed mainly of several stacks of saccules. The central core or medullary region contained numerous vesicular and tubular membranous profiles. Numerous cisternae of the endoplasmic reticulum (ER) were closely applied to the surface of the Golgi apparatus. In thin sections, the ER cisternae were separated from the stacks of saccules by small spherical or elongated membranous profiles. In thick sections, most of these elements formed a network of tubules, some of which being continuous with ER cisternae. Cisternae of the endoplasmic reticulum near the cis-face of the Golgi stacks also branched and traversed the cortical region of the Golgi apparatus through gaps seen between the stacks of saccules to reach the central core region. Some of these were closely applied to the trans elements of Golgi stacks although never in continuity with them. Finally, ER cisternae were found located within the stacks themselves between Golgi saccules; here the membranes of the closely apposed cisternae of the endoplasmic reticulum and Golgi saccules remained separated by a space of about 12 nm. Thus, in the spermatid, the endoplasmic reticulum was closely related with all components of the Golgi apparatus. Studies performed with the electron microscope have demonstrated that the Golgi apparatus of early spermatids of various mammals forms a compact hemispherical mass, next to the developing acrosomic system, and is composed of several peripherally located stacks of saccules enveloping a core region containing vesicular and tubular membranous profiles (Burgos and Fawcett, '55; Gardner, '66; Beams and Kessel, '68; Sandoz, '70; Susi et al., '71; Dooher and Bennett, '73; Mollenhauer et al., '76). Cisternae of the endoplasmic reticulum have been shown to be closely applied to the convex outer surface of the Golgi apparatus (Sandoz, '70; Susi et al., '71; Mollenhauer et al., '76; Clermont and Rambourg, '78). These cisternae were found to be separated from the stacks of saccules by a ANAT. REC. (1979) 193: 243-256.
layer, called the peripheral region of the Golgi apparatus, containing numerous small vesicles and tubules arranged in a haphazard manner, with the latter tubules being seemingly continuous with the saccules of the stacks (Susi et al., '71). In the following description, evidence will be presented indicating that in addition to being located on the cis-face (terminology of Ehrenreich et al., '73) of the Golgi apparatus, endoplasmic reticulum cisternae crossed the gaps between stacks of saccules and occupied the central core region. They were also inserted in the stacks themselves where they were sandwiched between Golgi saccules. Received June 15, '18.Accepted Sept. 9, '78. I Ddpartement de Biologie, Centre dEtudes Nucleaires de Saclay. B.P. No 2, 91 Gif-sup-Yvette, France.
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L. HERMO, Y. CLERMONT AND A. RAMBOURG MATERIALS AND METHODS
Testes of sexually mature albino rats were fixed by perfusion through the abdominal aorta according to t h e method of Vitale et al. ('73) with 2.5% or 5%glutaraldehyde buffered with either s-collidine (0.2 M) or sodium cacodylate (0.2 M). After 15 minutes of perfusion, they were removed from t h e scrotum, trimmed into small (1mm) cubes and placed in t h e same fixative for a n additional one hour. The tissue was then washed in t h e same buffer as used in the fixative and treated according to one of three staining procedures. In one procedure, t h e tissue was postfixed with 1%osmium tetroxide and then treated with tannic acid according to t h e method of Simionescu and Simionescu ('76). After osmication, the tissue was rinsed for 15 minutes in a 0.1 M sodium cacodylate buffer. It was then transferred for 30 minutes into a 1%solution of tannic acid in t h e same buffer and finally washed for five minutes in a 0.1 M sodium cacodylate buffer containing 1%sodium sulfate. In the second procedure, t h e tissue was postfixed on the following day for two hours at 4°C in ferrocyanide-reduced osmium (i.e., a mixture of aqueous solutions of 2!& osmium tetroxide and 3% potassium ferrocyanide in a 1:l ratio by volume) (Karnovsky, '71). In t h e third procedure, t h e tissue was impregnated according to t h e technique described by Thiery and Rambourg ('76) to be referred to in this study as t h e Ur-Pb-Cu method. After immersion in 5% uranyl acetate in distilled water for one hour at 40"C, t h e tissue was placed for one hour a t 40°C in a n aqueous solution of lead and copper citrate and then postfixed overnight at 4°C in 1%osmium tetroxide. After each one of these various procedures, the pieces of tissue were dehydrated in ethanol and then embedded in Epon. In t h e case of tissues treated with tannic acid or ferrocyanide-reduced osmium, thin sections of selected fields were cut, mounted on copper grids, stained with uranyl acetate and lead citrate and examined with a Philips 400 electron microscope at 80 or 60 kv. In the case of t h e UrPb-Cu-treated tissue, both thin and thick (0.25-0.5 pm) sections were cut, mounted on copper grids and examined without further staining. The thick sections were observed at 100 kv, and stereopairs were prepared by photographing t h e same field after tilting t h e specimen every 6" from t h e -45" position to the +45" position of t h e goniometric stage of the electron microscope. In this manner,
three-dimensional images could be obtained which helped clarify t h e structural relationship between the endoplasmic reticulum and t h e components of t h e Golgi apparatus. RESULTS
The Golgi apparatus of spermatids a t the Golgi and cap phases could be seen, when examined at low magnification with any one of t h e three staining procedures, to form a compact hemispherical mass whose base was located next to t h e developing acrosomic system (figs. 1, 2). While several stacks of saccules, separated from each other by narrow gaps, generally occupied t h e periphery or cortex of the hemisphere, t h e central core or medullary region contained numerous vesicular and tubular membranous profiles of various sizes and shapes (figs. 1, 2 ) . Between t h e cisternae of t h e endoplasmic reticulum (ER) and the cis-face of t h e Golgi stacks, a peripheral Golgi region characterized by a variable number of vesicular or tubular membranous profiles was also clearly visible (fig. 1). The endoplasmic reticulum of spermatids, heavily stained with t h e Ur-Pb-Cu technique, as already described by Clermont and Rambourg ('78),appeared in thick sections as a continuous system in which distended spherical or fusiform cisternae were connected by narrow bridges (fig. 7 ) . In t h e vicinity of the Golgi apparatus, flattened cisternae of the endoplasmic reticulum formed a tight network which capped t h e cis-face of the Golgi apparatus (figs. 2, 7 , 9). In thick sections of material treated with Ur-Pb-Cu, most of t h e membranous profiles seen between t h e cisternae of t h e endoplasmic reticulum and t h e stacks of saccules formed a n elaborate network of anastomotic tubules (figs. 2 , 9 ) ,with a few of these tubules appearing to be continuous with t h e surface of t h e ER cisternae facing t h e Golgi apparatus (fig. 2 ) . A close examination of such tubules in stereopairs taken every 6" from t h e -45" position to the t 4 5 " position of t h e goniometric stage did indeed confirm t h a t cisternae of t h e endoplasmic reticulum were connected with t h e underlying tubular network (fig. 8). In both thin (fig. 3) and thick (figs. 7-91 sections of t h e Ur-Pb-Cu impregnated tissue or thin sections of tissue treated with ferrocyanide-reduced osmium (fig. 4) or tannic acid (fig. 61, cisternae of t h e endoplasmic reticulum were also occasionally observed in t h e gaps between adjacent stacks of Golgi sac-
ER-GOLGI RELATIONS IN RAT SPERMATIDS
cules. These cisternae appeared as collateral branches of t h e ER cisternae capping the cisface of t h e Golgi apparatus (fig. 4) and ended in t h e central core region by running through t h e gaps (fig. 3). In t h e central core region, elements of the endoplasmic reticulum were easily identified in tissue impregnated with t h e Ur-Pb-Cu technique since they were more intensely stained t h a n t h e other tubular or vesicular elements present in this region. In tissues t r e a t e d w i t h ferrocyanide-reduced osmium or tannic acid, t h e identification of these elements of endoplasmic reticulum was more difficult, but they could be recognized by their similarity in size, shape and content to those ER cisternae distributed in the cytoplasm surrounding t h e Golgi apparatus. In t h e central core region, the cisternae of t h e endoplasmic reticulum were loosely interconnected with one another by narrow bridges and formed a continuous system (figs. 3 , 5 , 7). Some of t h e cisternae established a close association with this trans-face of t h e Golgi stacks. In such a case t h e cisternae of the endoplasmic reticulum always remained separated by a small intervening space from t h e trans element of t h e Golgi stack (figs. 3, 5, 6). This trans element was either similar to t h e other saccules of t h e stack or showed a wider intermembranous lumen. ER cisternae were also found to be sandwiched between t h e Golgi saccules at any level of t h e stacks at either their center or periphery (figs. 5, 6). As on t h e trans-face, these ER cisternae always remained separated from t h e Golgi saccules by a space of about 12 nm. DISCUSSION
I t is a common observation t h a t i n electron micrographs of various cell types, including spermatids, smooth-surfaced blebs emanate from t h e surface of t h e endoplasmic reticulum cisternae facing t h e cis-face of t h e Golgi apparatus (Caro and Palade, '64; Mollenhauer, '65; Friend, '65; Manton, '66; Flickinger, '69; Sandoz, '70; Susi et al., '71; Chretien, '72). Since these blebs resemble t h e small vesicular profiles encountered in thin sections between the endoplasmic reticulum and the cis-face of a Golgi stack of saccules, i t has been suggested t h a t such blebs give rise to the vesicles, called intermediate or transfer vesicles, which then travel toward t h e Golgi elements with which they would fuse to deliver both their content and membrane (Zeigel and Dalton, '62; Essner and Novikoff, '62; Novikoff and Shin, '64;
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Kessel, '71; Bracker e t al., '71; Chretien, '72; Morre and Mollenhauer, '74; Palade, '75). In thin sections of the Golgi and cap phase spermatids, numerous vesicular and elongated membranous profiles a r e seen in t h e space separating t h e endoplasmic reticulum from t h e stacks of saccules already referred to as t h e peripheral region of t h e Golgi apparatus by Susi et al. ('71). When t h e thickness of t h e section is increased, however, and t h e three-dimensional arrangement of t h e UrPb-Cu stained structures viewed in stereopairs, most of these elements form a network of tubules, some of which are continuous with t h e overlying cisternae or endoplasmic reticulum. In t h e case of spermatids, therefore, as in some plant cells (Bracker et al., '711, a structural continuity appears to exist between t h e endoplasmic reticulum and t h e elaborate t u bular network seen on the cis-face of the stacks of saccules. Another common feature of t h e Golgi apparatus in Golgi and cap phase spermatids is t h e presence of perforating branches of t h e endoplasmic reticulum t h a t traverse the gaps between t h e Golgi stacks to reach t h e medullary region. Similar observations have been recently reported by Novikoff e t al. ('77) in exocrine pancreatic cells, where ER elements located on t h e cis-face were found to cross passageways between t h e Golgi stacks to become continuous with GERL on t h e trans-face of t h e Golgi apparatus. Since enzyme histochemistry was not performed, it is difficult to decide whether or not t h e trans-Golgi elements as well a s t h e tubular and vesicular membranous profiles in t h e medulla described in our material belong to GERL. Nevertheless, no continuity was observed between t h e transGolgi saccules or t h e above profiles and t h e endoplasmic reticulum. This is in keeping with what has been described by Hand and Oliver ('77) in the acinar cells of the rat exorbital lacrimal gland. Although there is a close topographical relationship between the two organelles as they run parallel to each other, they a r e nevertheless always separated from each other by a space. Such a close relationship between the endoplasmic reticulum and Golgi apparatus is not restricted to its cis- and trans-faces. Indeed, ER cisternae are also found to be lodged between t h e Golgi saccules at any level of the stack. The space separating t h e two organelles in this location is of t h e same order of magnitude as t h a t a t t h e trans-face. Thus, al-
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L. HERMO, Y. CLERMONT AND A. RAMBOURG
though t h e endoplasmic reticulum in spermatids is closely associated with all components of the Golgi apparatus, a structural continuity between t h e two organelles is observed only on the cis-face of t h e Golgi apparatus. ACKNOWLEDGMENTS
This work done in t h e Department of Anatomy, McGill University, was supported by a grant from the Medical Research Council of Canada. The travels of t h e authors between t h e two laboratories were made possible through a France-Quebec exchange program. The assistance of Doctor Michael Lalli is acknowledged. The help of Doctor G. Bennett in t h e preparation of t h e manuscript was appreciated. LITERATURE CITED Beams, H. W., and R. G. Kessel 1968 The Golgi apparatus: structure and function. Int. Rev. Cytol., 23: 209-276. Bracker, C. E., S. N. Grove, C. E. Heintz and D. J. Morre 1971 Continuity between endomembrane components in hyphae of Pythium spp. Cytobiol., 4: 1-8. Burgos, M. H., and D. W. Fawcett 1955 Studies on the fine structure of the mammalian testis. I. Differentiation of spermatids in the cat Welrs dornesticai. J. Biophys. Cytol., 1: 287-300. Caro. L. G., and G. E. Palade 1964 Protein synthesis, storage and discharge in the pancreatic exocrine cell. tJ. Cell Biol., 20: 473~495. Chretien. M. 1972 Action de la testosterone sur la structure fine d’un effecteur: la glande sous-maxillaire de la souris mile. 11. Reaction des tubes secreteurs a l’injection de testosterone chez le castrat. J. Microscopie, 14: 55-74. Clermont, Y., and A. Rambourg 1978 Evolution of the endoplasmic reticulum during r a t spermiogenesis. Am. J . Anat., 151: 191-212. Dooher, G. B., and D. Bennett 1973 Fine structural observations on the development of the sperm head in the mouse. Am. J. Anat., 136: 339-362. Ehrenreich. J. H., J. J. M. Bergeron, P. Siekevitz and G. E. Palade 1973 Golgi fractions prepared from rat liver homogenates. I. Isolation procedure and morphological characterization. J. Cell Biol.. 59: 45-72. Essner. E.. and A. B. Novikoff 1962 Cytological studies on two functional hepatomas. Interrelations of endoplasmlc reticulum, Golgi apparatus and lysosomes. J. Cell Biol., 15: 289-312. Flickinger, C. J. 1969 The pattern of growth of the Golgi complex during the fetal and postnatal development of rat epididymis. J. Ultrastr. Res., 27: 344-360. Friend, D. S. 1965 The fine structure of Brunner’s glands in the mouse. J. Cell Biol., 25: 563-576.
Gardner, P. J. 1966 Fine structure of the seminiferous tubule of the Swiss mouse. The spermatid. Anat. Rec., 155: 235-250. Hand, A. R., and C. Oliver 1977 Relationship between the Golgi apparatus, GERL, and secretory granules in acinar cells of the rat exorbital lacrimal gland. J. Cell Biol., 74: 399-413. Karnovsky, M. J. 1971 Use of ferrocyanide-reduced osmium tetroxide in electron microscopy. Proc. of Eleventh Ann. SOC.of Cell Biol., Nov. 17-20, 1971, New Orleans, Louisiana. Abstract 284, p. 146. Kessel, R. G. 1971 Origin of the Golgi apparatus in embryonic cells of the grasshopper. J. Ultrastr. Res., 34: 260-275. Manton, I. 1966 Observations on scale production in pyranomas amylifera Conrad. J. Cell. Sc., I : 429-438. Mollenhauer, H. H. 1965 Transition forms of Golg~apparatus secretion vesicles. J. Ultrastr. Res., 12: 439-446. Mollenhauer, H. H., B. S. Hass and D. J. Morre 1976 Membrane transformations in Golgi apparatus of rat spermatids. A role for thick cisternae and two classes of coated vesicles in acrosome formation. J. Microscopie. Biol. Cell., 27: 33-36. Morre, D. J., and H. H. Mollenhauer 1974 The endomemhrane concept: a functional integration of endoplasmic reticulum and Golgi apparatus. In: Dynamic Aspects of Plant Ultrastructure. A. E. Robards. ed. McGraw-Hill (U.K.), pp. 84-137. Novikoff, A. B., M. Mori, N. Quintana and A. Yam 1977 Studies on t h e secretory process in the mammalian exocrine pancreas. I. The condensing vacuoles. J. Cell Biol., 75: 148-165. Novikoff, A. B., and W. Y. Shin 1964 The endoplasmic reticulum in the Golgi zone and its relations to microbodies, Golgi apparatus and autophagic vacuoles in rat liver cells. J. Microscopie, 3: 187-206. Palade, G. 1975 Intracellular aspects of the process of protein synthesis. Science, 189: 347-358. Sandoz, D. 1970 Evolution des ultrastructures a u cours de la formation de l’acrosome du spermatozoide chez la souris. J. Microscopie., 9: 535-558. Simionescu, N., and M. Simionescu 1976 Galloylglucoses of low molecular weight as mordant in electron microscopy. I. Procedure and evidence for mordanting effect. J. Cell Biol., 70: 608-621. Susi, F. R., C. P. Leblond and Y. Clermont 1971 Changes in the Golgi apparatus during spermiogenesis in the rat. Am. J. Anat., 130: 251-278. Thiery, G., and A. Rambourg 1976 A new staining technique for studying thick sections in the electron microscope. J. Microscopie. Biol. Cell., 26: 103-106. Vitale, R., D. W. Fawcett and M. Dym 1973 The normal development of the blood-testis barrier and t h e effects of clomiphene and estrogen treatment. Anat. Rec., 176: 333-344. Zeigel, R. F., and A. J. Dalton 1962 Speculations based on the morphology of t h e Golgi system in several types of protein secreting cells. J. Cell Biol., 15: 45-54.
PLATES
PLATE 1 EXPLANATION OF FIGURES
1 Cap phase spermatid as seen in a thin section stained with ferrocyanide-reduced osmium. The Golgi apparatus forms a compact hemispherical mass located over the developing acrosomic system (A) closely applied to the surface of the nucleus ( N ) . The periphery of the hemisphere or cortical region is made up by several stacks of saccules ( S ) separated by gaps (GI, while the central core or medullary region (MI contains vesicular and tubular membranous profiles. On the convex or cis-face of the Golgi apparatus, elongated cisternae of the endoplasmic reticulum (ER) cap the stacks of saccules from which they are separated by a space containing vesicular or tubular profiles. X 30,625. 2 Cap phase spermatid as seen in a 0.25 Fm thick section stained with the Ur-Pb-Cu technique. The head cap of the acrosomic system (A), applied to the nucleus (N) is closely associated with the hemispherical Golgi apparatus. In this section, the two regions of the Golgi apparatus, i.e., the cortical and the medullary regions are well delineated. In close relation with the outer surface of the cortical region consisting of the stacks of saccules ( S ) , are elongated cisternae of the endoplasmic reticulum (ER). In the medullary region the heavily impregnated ER elements (ER) are easily distinguished from poorly stained spherical or tubular structures. X 29,250.
248
ER-GOl.(;I RELATIONS IN RAT SPERMATIDS L Hermo. Y Clermont and A Rambourg
PLATE 1
249
PLATE 2
250
:3
Golgi phase spermatid as seen in thin section stained with t h e Ur-Pb-Cu technique. Cisternae of t h e endoplasmic reticulum lER1 a r e evident in t h e surrounding cyto plasm and over the cis-face of t h e Go1g-1 apparatus. The stacks of saccules (SI of the cortical remon of t h e Golgi apparatus are separated from each other by gaps ((;I containing vesicles a n d tubules. A cisterna of t h e endoplasmic reticulum is seen to traverse one of these gaps, reach t h e medullary central core reDon (MI and apply itself to t h e t r a n s element of t h e G o l g stack (arrowheads). In t h e central core repion. the heavily impregnated endoplasmic reticulum (ERI is also found loosely d i s ~ tributed among pale spherical or ovoid membranous profiles. Another cisterna of the endoplasmic reticulum is closely associated with t h e t r a n s element of t h e G o l g ~ stack. hut a uniform space of about 1 2 nm separates t h e two organelles from each other (vertical arrow). ( A ) acrosomic granule of t h e acrosomic system. 37.000
4
Portion of t h e G o l g ~apparatus of a Golgi phase spermatid in a thin section stained with ferrocyanide~reducedosmium. At the periphery of t h e Golg.1 apparatus there are several cisternae of t h e endoplasmic reticulum (ERI which cap t h e cis-face N o t e t h a t these elements are similar in size. shape and content t o those cisternal elements of t h e endoplasmic reticulum seen in t h e surrounding cytoplasm. In one area. an EK cisterna over t h e cis-face can be seen t o form a branch (arrowhead, which penetrates through t h e gap between adjacent stacks of saccules is1 in t h e direction of t h e medullary r e p o n ( M I . In t h e latter location. tubular and vesicular membranous profiles are present and a proacrosomic granule (Pi. x 42,000.
ER-GOLGI RELATIONS IN R A T SPERMATIDS L Hermo. Y Clermont and A Rambourg
PLATE 2
PLATE 3 EXPLANATION OF FIGURES
5
Cap phase spermatid as seen in a thin section stained with ferrocyanide-reduced osmium. The cortical region of the Golgi apparatus shows a stack consisting of several saccules ( 3 .A few ER elements (ER) are seen over the cis-face and one can also be found in t h e gap between stacks (arrowhead). Elements of t h e endoplasmic reticulum are also found free i n the medullary region (MI. In addition, a cisterna of the endoplasmic reticulum is also found to be directly lodged within the stack of saccules (asterisk). Note the similarity in appearance between this cisterna of ER and those seen elsewhere. I n this latter location, two Golgi saccules diverge from each other to enclose the ER cisterna within the stack, but no direct sign of communication between t h e two organelles is evident. Also labeled: acrosomic system ( A ) , nucleus (N). X 50,000.
6 Cap phase spermatid as seen in a thin section treated with tannic acid. In this type of preparation, the content of the endoplasmic reticulum is well stained and readily apparent, as can be seen for those cisternae in the surrounding cytoplasm. Similar cisternal elements of the endoplasmic reticulum (ER) can also be seen overlying the cis-face of t h e Golgi apparatus as well as in the medullary reQon. One cistema of the endoplasmic reticulum (asterisk), similar in appearance to those seen elsewhere. IS sandwiched between the Go]@ saccules of a stack. This cisterna is completely ~ but nevertheless separated from them by enveloped by the adjacent G o l saccules, a space of about 12 nm. The empty looking membranous profiles marked “ X ’ cannot be identified. Also labeled: head cap of acrosomic system (A), nucleus (N). X 50,000.
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ER-GOLGI RELATIONS IN RAT SPERMATIDS L Herrno. Y Clermont a n d A Rambourg
PLATE 4 EXPLANATION OF FIGURES
Figs. 7-9 The photographs of this plate a r e arranged in pairs. The two members of each pair a r e photographs of t h e same field t a k e n at different angles (e.g., -6" and 1 6 " ) of t h e goniometric stage of t h e electron microscope. From t h e two pictures of t h e pair or stereopair, a single stereoscopic image can be obtained by using a stereoscopic binocular lens. The section was stained with t h e Ur-Pb-Cu technique.
7 Cap phase spermatid a s seen in a 0.5 p m thick section stained with t h e Ur-Pb-Cu technique Thm relatively thick section shows t h e structural complexity of t h e Golgi apparatus. At t h e periphery or in the central core r e g o n t h e ER cisternae a r e interconnected by narrow bridges. ER cisternae a r e closely apposed t o t h e cis-face of t h e Golgi apparatus. S, stack of saccules; N, nucleus; A, acrosomic system. X 15,000. 8 Cap phase spermatid already seen in figure 2 but shown here in a stereopair t o illustrate t h e relationship in three-dimensions, of t h e ER cisternae with t h e underlying elements of t h e Golgi apparatus. On t h e convex surface of t h e Golgi apparatus. ER cisternae a r e continuous with tubular elements seen between the cisternae and t h e G o l g stacks. In t h e cortical zone, t h e stacks of saccules a r e interrupted by gaps. In the central core region, cisternae of t h e endoplasmic reticulum a r e labeled ER. S, stack of saccules; N, nucleus, A , acrosomic system. Section thickness, 0.25 pm. X 15,200.
9 Cap phase spermatid a s seen in a 0.25 pm thick section stained with t h e Ur-Pb-Cu technique. ER elements ( E R ) present a t t h e periphery of t h e Golgi mass a s seen in a gap between Golgi stacks (vertical arrow) Er cisternae a r e seen in the medullary region (arrowheads) and one cisterna is closely associated to a Golgi element located on t h e trans-face of a stack (oblique arrow). S, stack of saccules; N , nucleus: A, acrosomic system. x 15,200.
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ER-GOLGI RELATIONS IN RAT SPERMATIDS L. Hermo, Y. Clermont and A. Rambourg
PLATE 4
ABSTRACT The appearance of the spermatid Golgi apparatus was studied, both in thin sections of rat testes fixed in glutaraldehyde and treated with either tannic acid or ferrocyanide-reduced osmium, and in relatively thick sections (0.25-0.5pm) of glutaraldehyde fixed tissue impregnated with uranyl acetate followed by lead and copper citrate. With any one of these three procedures, the Golgi apparatus of young spermatids examined a t the Golgi and cap phases appeared as a compact hemispherical mass whose base was located next to the developing acrosomic system. The cortical region of the hemisphere was composed mainly of several stacks of saccules. The central core or medullary region contained numerous vesicular and tubular membranous profiles. Numerous cisternae of the endoplasmic reticulum (ER) were closely applied to the surface of the Golgi apparatus. In thin sections, the ER cisternae were separated from the stacks of saccules by small spherical or elongated membranous profiles. In thick sections, most of these elements formed a network of tubules, some of which being continuous with ER cisternae. Cisternae of the endoplasmic reticulum near the cis-face of the Golgi stacks also branched and traversed the cortical region of the Golgi apparatus through gaps seen between the stacks of saccules to reach the central core region. Some of these were closely applied to the trans elements of Golgi stacks although never in continuity with them. Finally, ER cisternae were found located within the stacks themselves between Golgi saccules; here the membranes of the closely apposed cisternae of the endoplasmic reticulum and Golgi saccules remained separated by a space of about 12 nm. Thus, in the spermatid, the endoplasmic reticulum was closely related with all components of the Golgi apparatus. Studies performed with the electron microscope have demonstrated that the Golgi apparatus of early spermatids of various mammals forms a compact hemispherical mass, next to the developing acrosomic system, and is composed of several peripherally located stacks of saccules enveloping a core region containing vesicular and tubular membranous profiles (Burgos and Fawcett, '55; Gardner, '66; Beams and Kessel, '68; Sandoz, '70; Susi et al., '71; Dooher and Bennett, '73; Mollenhauer et al., '76). Cisternae of the endoplasmic reticulum have been shown to be closely applied to the convex outer surface of the Golgi apparatus (Sandoz, '70; Susi et al., '71; Mollenhauer et al., '76; Clermont and Rambourg, '78). These cisternae were found to be separated from the stacks of saccules by a ANAT. REC. (1979) 193: 243-256.
layer, called the peripheral region of the Golgi apparatus, containing numerous small vesicles and tubules arranged in a haphazard manner, with the latter tubules being seemingly continuous with the saccules of the stacks (Susi et al., '71). In the following description, evidence will be presented indicating that in addition to being located on the cis-face (terminology of Ehrenreich et al., '73) of the Golgi apparatus, endoplasmic reticulum cisternae crossed the gaps between stacks of saccules and occupied the central core region. They were also inserted in the stacks themselves where they were sandwiched between Golgi saccules. Received June 15, '18.Accepted Sept. 9, '78. I Ddpartement de Biologie, Centre dEtudes Nucleaires de Saclay. B.P. No 2, 91 Gif-sup-Yvette, France.
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L. HERMO, Y. CLERMONT AND A. RAMBOURG MATERIALS AND METHODS
Testes of sexually mature albino rats were fixed by perfusion through the abdominal aorta according to t h e method of Vitale et al. ('73) with 2.5% or 5%glutaraldehyde buffered with either s-collidine (0.2 M) or sodium cacodylate (0.2 M). After 15 minutes of perfusion, they were removed from t h e scrotum, trimmed into small (1mm) cubes and placed in t h e same fixative for a n additional one hour. The tissue was then washed in t h e same buffer as used in the fixative and treated according to one of three staining procedures. In one procedure, t h e tissue was postfixed with 1%osmium tetroxide and then treated with tannic acid according to t h e method of Simionescu and Simionescu ('76). After osmication, the tissue was rinsed for 15 minutes in a 0.1 M sodium cacodylate buffer. It was then transferred for 30 minutes into a 1%solution of tannic acid in t h e same buffer and finally washed for five minutes in a 0.1 M sodium cacodylate buffer containing 1%sodium sulfate. In the second procedure, t h e tissue was postfixed on the following day for two hours at 4°C in ferrocyanide-reduced osmium (i.e., a mixture of aqueous solutions of 2!& osmium tetroxide and 3% potassium ferrocyanide in a 1:l ratio by volume) (Karnovsky, '71). In t h e third procedure, t h e tissue was impregnated according to t h e technique described by Thiery and Rambourg ('76) to be referred to in this study as t h e Ur-Pb-Cu method. After immersion in 5% uranyl acetate in distilled water for one hour at 40"C, t h e tissue was placed for one hour a t 40°C in a n aqueous solution of lead and copper citrate and then postfixed overnight at 4°C in 1%osmium tetroxide. After each one of these various procedures, the pieces of tissue were dehydrated in ethanol and then embedded in Epon. In t h e case of tissues treated with tannic acid or ferrocyanide-reduced osmium, thin sections of selected fields were cut, mounted on copper grids, stained with uranyl acetate and lead citrate and examined with a Philips 400 electron microscope at 80 or 60 kv. In the case of t h e UrPb-Cu-treated tissue, both thin and thick (0.25-0.5 pm) sections were cut, mounted on copper grids and examined without further staining. The thick sections were observed at 100 kv, and stereopairs were prepared by photographing t h e same field after tilting t h e specimen every 6" from t h e -45" position to the +45" position of t h e goniometric stage of the electron microscope. In this manner,
three-dimensional images could be obtained which helped clarify t h e structural relationship between the endoplasmic reticulum and t h e components of t h e Golgi apparatus. RESULTS
The Golgi apparatus of spermatids a t the Golgi and cap phases could be seen, when examined at low magnification with any one of t h e three staining procedures, to form a compact hemispherical mass whose base was located next to t h e developing acrosomic system (figs. 1, 2). While several stacks of saccules, separated from each other by narrow gaps, generally occupied t h e periphery or cortex of the hemisphere, t h e central core or medullary region contained numerous vesicular and tubular membranous profiles of various sizes and shapes (figs. 1, 2 ) . Between t h e cisternae of t h e endoplasmic reticulum (ER) and the cis-face of t h e Golgi stacks, a peripheral Golgi region characterized by a variable number of vesicular or tubular membranous profiles was also clearly visible (fig. 1). The endoplasmic reticulum of spermatids, heavily stained with t h e Ur-Pb-Cu technique, as already described by Clermont and Rambourg ('78),appeared in thick sections as a continuous system in which distended spherical or fusiform cisternae were connected by narrow bridges (fig. 7 ) . In t h e vicinity of the Golgi apparatus, flattened cisternae of the endoplasmic reticulum formed a tight network which capped t h e cis-face of the Golgi apparatus (figs. 2, 7 , 9). In thick sections of material treated with Ur-Pb-Cu, most of t h e membranous profiles seen between t h e cisternae of t h e endoplasmic reticulum and t h e stacks of saccules formed a n elaborate network of anastomotic tubules (figs. 2 , 9 ) ,with a few of these tubules appearing to be continuous with t h e surface of t h e ER cisternae facing t h e Golgi apparatus (fig. 2 ) . A close examination of such tubules in stereopairs taken every 6" from t h e -45" position to the t 4 5 " position of t h e goniometric stage did indeed confirm t h a t cisternae of t h e endoplasmic reticulum were connected with t h e underlying tubular network (fig. 8). In both thin (fig. 3) and thick (figs. 7-91 sections of t h e Ur-Pb-Cu impregnated tissue or thin sections of tissue treated with ferrocyanide-reduced osmium (fig. 4) or tannic acid (fig. 61, cisternae of t h e endoplasmic reticulum were also occasionally observed in t h e gaps between adjacent stacks of Golgi sac-
ER-GOLGI RELATIONS IN RAT SPERMATIDS
cules. These cisternae appeared as collateral branches of t h e ER cisternae capping the cisface of t h e Golgi apparatus (fig. 4) and ended in t h e central core region by running through t h e gaps (fig. 3). In t h e central core region, elements of the endoplasmic reticulum were easily identified in tissue impregnated with t h e Ur-Pb-Cu technique since they were more intensely stained t h a n t h e other tubular or vesicular elements present in this region. In tissues t r e a t e d w i t h ferrocyanide-reduced osmium or tannic acid, t h e identification of these elements of endoplasmic reticulum was more difficult, but they could be recognized by their similarity in size, shape and content to those ER cisternae distributed in the cytoplasm surrounding t h e Golgi apparatus. In t h e central core region, the cisternae of t h e endoplasmic reticulum were loosely interconnected with one another by narrow bridges and formed a continuous system (figs. 3 , 5 , 7). Some of t h e cisternae established a close association with this trans-face of t h e Golgi stacks. In such a case t h e cisternae of the endoplasmic reticulum always remained separated by a small intervening space from t h e trans element of t h e Golgi stack (figs. 3, 5, 6). This trans element was either similar to t h e other saccules of t h e stack or showed a wider intermembranous lumen. ER cisternae were also found to be sandwiched between t h e Golgi saccules at any level of t h e stacks at either their center or periphery (figs. 5, 6). As on t h e trans-face, these ER cisternae always remained separated from t h e Golgi saccules by a space of about 12 nm. DISCUSSION
I t is a common observation t h a t i n electron micrographs of various cell types, including spermatids, smooth-surfaced blebs emanate from t h e surface of t h e endoplasmic reticulum cisternae facing t h e cis-face of t h e Golgi apparatus (Caro and Palade, '64; Mollenhauer, '65; Friend, '65; Manton, '66; Flickinger, '69; Sandoz, '70; Susi et al., '71; Chretien, '72). Since these blebs resemble t h e small vesicular profiles encountered in thin sections between the endoplasmic reticulum and the cis-face of a Golgi stack of saccules, i t has been suggested t h a t such blebs give rise to the vesicles, called intermediate or transfer vesicles, which then travel toward t h e Golgi elements with which they would fuse to deliver both their content and membrane (Zeigel and Dalton, '62; Essner and Novikoff, '62; Novikoff and Shin, '64;
245
Kessel, '71; Bracker e t al., '71; Chretien, '72; Morre and Mollenhauer, '74; Palade, '75). In thin sections of the Golgi and cap phase spermatids, numerous vesicular and elongated membranous profiles a r e seen in t h e space separating t h e endoplasmic reticulum from t h e stacks of saccules already referred to as t h e peripheral region of t h e Golgi apparatus by Susi et al. ('71). When t h e thickness of t h e section is increased, however, and t h e three-dimensional arrangement of t h e UrPb-Cu stained structures viewed in stereopairs, most of these elements form a network of tubules, some of which are continuous with t h e overlying cisternae or endoplasmic reticulum. In t h e case of spermatids, therefore, as in some plant cells (Bracker et al., '711, a structural continuity appears to exist between t h e endoplasmic reticulum and t h e elaborate t u bular network seen on the cis-face of the stacks of saccules. Another common feature of t h e Golgi apparatus in Golgi and cap phase spermatids is t h e presence of perforating branches of t h e endoplasmic reticulum t h a t traverse the gaps between t h e Golgi stacks to reach t h e medullary region. Similar observations have been recently reported by Novikoff e t al. ('77) in exocrine pancreatic cells, where ER elements located on t h e cis-face were found to cross passageways between t h e Golgi stacks to become continuous with GERL on t h e trans-face of t h e Golgi apparatus. Since enzyme histochemistry was not performed, it is difficult to decide whether or not t h e trans-Golgi elements as well a s t h e tubular and vesicular membranous profiles in t h e medulla described in our material belong to GERL. Nevertheless, no continuity was observed between t h e transGolgi saccules or t h e above profiles and t h e endoplasmic reticulum. This is in keeping with what has been described by Hand and Oliver ('77) in the acinar cells of the rat exorbital lacrimal gland. Although there is a close topographical relationship between the two organelles as they run parallel to each other, they a r e nevertheless always separated from each other by a space. Such a close relationship between the endoplasmic reticulum and Golgi apparatus is not restricted to its cis- and trans-faces. Indeed, ER cisternae are also found to be lodged between t h e Golgi saccules at any level of the stack. The space separating t h e two organelles in this location is of t h e same order of magnitude as t h a t a t t h e trans-face. Thus, al-
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L. HERMO, Y. CLERMONT AND A. RAMBOURG
though t h e endoplasmic reticulum in spermatids is closely associated with all components of the Golgi apparatus, a structural continuity between t h e two organelles is observed only on the cis-face of t h e Golgi apparatus. ACKNOWLEDGMENTS
This work done in t h e Department of Anatomy, McGill University, was supported by a grant from the Medical Research Council of Canada. The travels of t h e authors between t h e two laboratories were made possible through a France-Quebec exchange program. The assistance of Doctor Michael Lalli is acknowledged. The help of Doctor G. Bennett in t h e preparation of t h e manuscript was appreciated. LITERATURE CITED Beams, H. W., and R. G. Kessel 1968 The Golgi apparatus: structure and function. Int. Rev. Cytol., 23: 209-276. Bracker, C. E., S. N. Grove, C. E. Heintz and D. J. Morre 1971 Continuity between endomembrane components in hyphae of Pythium spp. Cytobiol., 4: 1-8. Burgos, M. H., and D. W. Fawcett 1955 Studies on the fine structure of the mammalian testis. I. Differentiation of spermatids in the cat Welrs dornesticai. J. Biophys. Cytol., 1: 287-300. Caro. L. G., and G. E. Palade 1964 Protein synthesis, storage and discharge in the pancreatic exocrine cell. tJ. Cell Biol., 20: 473~495. Chretien. M. 1972 Action de la testosterone sur la structure fine d’un effecteur: la glande sous-maxillaire de la souris mile. 11. Reaction des tubes secreteurs a l’injection de testosterone chez le castrat. J. Microscopie, 14: 55-74. Clermont, Y., and A. Rambourg 1978 Evolution of the endoplasmic reticulum during r a t spermiogenesis. Am. J . Anat., 151: 191-212. Dooher, G. B., and D. Bennett 1973 Fine structural observations on the development of the sperm head in the mouse. Am. J. Anat., 136: 339-362. Ehrenreich. J. H., J. J. M. Bergeron, P. Siekevitz and G. E. Palade 1973 Golgi fractions prepared from rat liver homogenates. I. Isolation procedure and morphological characterization. J. Cell Biol.. 59: 45-72. Essner. E.. and A. B. Novikoff 1962 Cytological studies on two functional hepatomas. Interrelations of endoplasmlc reticulum, Golgi apparatus and lysosomes. J. Cell Biol., 15: 289-312. Flickinger, C. J. 1969 The pattern of growth of the Golgi complex during the fetal and postnatal development of rat epididymis. J. Ultrastr. Res., 27: 344-360. Friend, D. S. 1965 The fine structure of Brunner’s glands in the mouse. J. Cell Biol., 25: 563-576.
Gardner, P. J. 1966 Fine structure of the seminiferous tubule of the Swiss mouse. The spermatid. Anat. Rec., 155: 235-250. Hand, A. R., and C. Oliver 1977 Relationship between the Golgi apparatus, GERL, and secretory granules in acinar cells of the rat exorbital lacrimal gland. J. Cell Biol., 74: 399-413. Karnovsky, M. J. 1971 Use of ferrocyanide-reduced osmium tetroxide in electron microscopy. Proc. of Eleventh Ann. SOC.of Cell Biol., Nov. 17-20, 1971, New Orleans, Louisiana. Abstract 284, p. 146. Kessel, R. G. 1971 Origin of the Golgi apparatus in embryonic cells of the grasshopper. J. Ultrastr. Res., 34: 260-275. Manton, I. 1966 Observations on scale production in pyranomas amylifera Conrad. J. Cell. Sc., I : 429-438. Mollenhauer, H. H. 1965 Transition forms of Golg~apparatus secretion vesicles. J. Ultrastr. Res., 12: 439-446. Mollenhauer, H. H., B. S. Hass and D. J. Morre 1976 Membrane transformations in Golgi apparatus of rat spermatids. A role for thick cisternae and two classes of coated vesicles in acrosome formation. J. Microscopie. Biol. Cell., 27: 33-36. Morre, D. J., and H. H. Mollenhauer 1974 The endomemhrane concept: a functional integration of endoplasmic reticulum and Golgi apparatus. In: Dynamic Aspects of Plant Ultrastructure. A. E. Robards. ed. McGraw-Hill (U.K.), pp. 84-137. Novikoff, A. B., M. Mori, N. Quintana and A. Yam 1977 Studies on t h e secretory process in the mammalian exocrine pancreas. I. The condensing vacuoles. J. Cell Biol., 75: 148-165. Novikoff, A. B., and W. Y. Shin 1964 The endoplasmic reticulum in the Golgi zone and its relations to microbodies, Golgi apparatus and autophagic vacuoles in rat liver cells. J. Microscopie, 3: 187-206. Palade, G. 1975 Intracellular aspects of the process of protein synthesis. Science, 189: 347-358. Sandoz, D. 1970 Evolution des ultrastructures a u cours de la formation de l’acrosome du spermatozoide chez la souris. J. Microscopie., 9: 535-558. Simionescu, N., and M. Simionescu 1976 Galloylglucoses of low molecular weight as mordant in electron microscopy. I. Procedure and evidence for mordanting effect. J. Cell Biol., 70: 608-621. Susi, F. R., C. P. Leblond and Y. Clermont 1971 Changes in the Golgi apparatus during spermiogenesis in the rat. Am. J. Anat., 130: 251-278. Thiery, G., and A. Rambourg 1976 A new staining technique for studying thick sections in the electron microscope. J. Microscopie. Biol. Cell., 26: 103-106. Vitale, R., D. W. Fawcett and M. Dym 1973 The normal development of the blood-testis barrier and t h e effects of clomiphene and estrogen treatment. Anat. Rec., 176: 333-344. Zeigel, R. F., and A. J. Dalton 1962 Speculations based on the morphology of t h e Golgi system in several types of protein secreting cells. J. Cell Biol., 15: 45-54.
PLATES
PLATE 1 EXPLANATION OF FIGURES
1 Cap phase spermatid as seen in a thin section stained with ferrocyanide-reduced osmium. The Golgi apparatus forms a compact hemispherical mass located over the developing acrosomic system (A) closely applied to the surface of the nucleus ( N ) . The periphery of the hemisphere or cortical region is made up by several stacks of saccules ( S ) separated by gaps (GI, while the central core or medullary region (MI contains vesicular and tubular membranous profiles. On the convex or cis-face of the Golgi apparatus, elongated cisternae of the endoplasmic reticulum (ER) cap the stacks of saccules from which they are separated by a space containing vesicular or tubular profiles. X 30,625. 2 Cap phase spermatid as seen in a 0.25 Fm thick section stained with the Ur-Pb-Cu technique. The head cap of the acrosomic system (A), applied to the nucleus (N) is closely associated with the hemispherical Golgi apparatus. In this section, the two regions of the Golgi apparatus, i.e., the cortical and the medullary regions are well delineated. In close relation with the outer surface of the cortical region consisting of the stacks of saccules ( S ) , are elongated cisternae of the endoplasmic reticulum (ER). In the medullary region the heavily impregnated ER elements (ER) are easily distinguished from poorly stained spherical or tubular structures. X 29,250.
248
ER-GOl.(;I RELATIONS IN RAT SPERMATIDS L Hermo. Y Clermont and A Rambourg
PLATE 1
249
PLATE 2
250
:3
Golgi phase spermatid as seen in thin section stained with t h e Ur-Pb-Cu technique. Cisternae of t h e endoplasmic reticulum lER1 a r e evident in t h e surrounding cyto plasm and over the cis-face of t h e Go1g-1 apparatus. The stacks of saccules (SI of the cortical remon of t h e Golgi apparatus are separated from each other by gaps ((;I containing vesicles a n d tubules. A cisterna of t h e endoplasmic reticulum is seen to traverse one of these gaps, reach t h e medullary central core reDon (MI and apply itself to t h e t r a n s element of t h e G o l g stack (arrowheads). In t h e central core repion. the heavily impregnated endoplasmic reticulum (ERI is also found loosely d i s ~ tributed among pale spherical or ovoid membranous profiles. Another cisterna of the endoplasmic reticulum is closely associated with t h e t r a n s element of t h e G o l g ~ stack. hut a uniform space of about 1 2 nm separates t h e two organelles from each other (vertical arrow). ( A ) acrosomic granule of t h e acrosomic system. 37.000
4
Portion of t h e G o l g ~apparatus of a Golgi phase spermatid in a thin section stained with ferrocyanide~reducedosmium. At the periphery of t h e Golg.1 apparatus there are several cisternae of t h e endoplasmic reticulum (ERI which cap t h e cis-face N o t e t h a t these elements are similar in size. shape and content t o those cisternal elements of t h e endoplasmic reticulum seen in t h e surrounding cytoplasm. In one area. an EK cisterna over t h e cis-face can be seen t o form a branch (arrowhead, which penetrates through t h e gap between adjacent stacks of saccules is1 in t h e direction of t h e medullary r e p o n ( M I . In t h e latter location. tubular and vesicular membranous profiles are present and a proacrosomic granule (Pi. x 42,000.
ER-GOLGI RELATIONS IN R A T SPERMATIDS L Hermo. Y Clermont and A Rambourg
PLATE 2
PLATE 3 EXPLANATION OF FIGURES
5
Cap phase spermatid as seen in a thin section stained with ferrocyanide-reduced osmium. The cortical region of the Golgi apparatus shows a stack consisting of several saccules ( 3 .A few ER elements (ER) are seen over the cis-face and one can also be found in t h e gap between stacks (arrowhead). Elements of t h e endoplasmic reticulum are also found free i n the medullary region (MI. In addition, a cisterna of the endoplasmic reticulum is also found to be directly lodged within the stack of saccules (asterisk). Note the similarity in appearance between this cisterna of ER and those seen elsewhere. I n this latter location, two Golgi saccules diverge from each other to enclose the ER cisterna within the stack, but no direct sign of communication between t h e two organelles is evident. Also labeled: acrosomic system ( A ) , nucleus (N). X 50,000.
6 Cap phase spermatid as seen in a thin section treated with tannic acid. In this type of preparation, the content of the endoplasmic reticulum is well stained and readily apparent, as can be seen for those cisternae in the surrounding cytoplasm. Similar cisternal elements of the endoplasmic reticulum (ER) can also be seen overlying the cis-face of t h e Golgi apparatus as well as in the medullary reQon. One cistema of the endoplasmic reticulum (asterisk), similar in appearance to those seen elsewhere. IS sandwiched between the Go]@ saccules of a stack. This cisterna is completely ~ but nevertheless separated from them by enveloped by the adjacent G o l saccules, a space of about 12 nm. The empty looking membranous profiles marked “ X ’ cannot be identified. Also labeled: head cap of acrosomic system (A), nucleus (N). X 50,000.
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ER-GOLGI RELATIONS IN RAT SPERMATIDS L Herrno. Y Clermont a n d A Rambourg
PLATE 4 EXPLANATION OF FIGURES
Figs. 7-9 The photographs of this plate a r e arranged in pairs. The two members of each pair a r e photographs of t h e same field t a k e n at different angles (e.g., -6" and 1 6 " ) of t h e goniometric stage of t h e electron microscope. From t h e two pictures of t h e pair or stereopair, a single stereoscopic image can be obtained by using a stereoscopic binocular lens. The section was stained with t h e Ur-Pb-Cu technique.
7 Cap phase spermatid a s seen in a 0.5 p m thick section stained with t h e Ur-Pb-Cu technique Thm relatively thick section shows t h e structural complexity of t h e Golgi apparatus. At t h e periphery or in the central core r e g o n t h e ER cisternae a r e interconnected by narrow bridges. ER cisternae a r e closely apposed t o t h e cis-face of t h e Golgi apparatus. S, stack of saccules; N, nucleus; A, acrosomic system. X 15,000. 8 Cap phase spermatid already seen in figure 2 but shown here in a stereopair t o illustrate t h e relationship in three-dimensions, of t h e ER cisternae with t h e underlying elements of t h e Golgi apparatus. On t h e convex surface of t h e Golgi apparatus. ER cisternae a r e continuous with tubular elements seen between the cisternae and t h e G o l g stacks. In t h e cortical zone, t h e stacks of saccules a r e interrupted by gaps. In the central core region, cisternae of t h e endoplasmic reticulum a r e labeled ER. S, stack of saccules; N, nucleus, A , acrosomic system. Section thickness, 0.25 pm. X 15,200.
9 Cap phase spermatid a s seen in a 0.25 pm thick section stained with t h e Ur-Pb-Cu technique. ER elements ( E R ) present a t t h e periphery of t h e Golgi mass a s seen in a gap between Golgi stacks (vertical arrow) Er cisternae a r e seen in the medullary region (arrowheads) and one cisterna is closely associated to a Golgi element located on t h e trans-face of a stack (oblique arrow). S, stack of saccules; N , nucleus: A, acrosomic system. x 15,200.
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ER-GOLGI RELATIONS IN RAT SPERMATIDS L. Hermo, Y. Clermont and A. Rambourg
PLATE 4
