THE ANATOMICAL RECORD 229:16-26 (1991)
The Golgi Apparatus of Rat Pachytene Spermatocytes During Spermatogenesis CARLOS A. SUAREZ-QUIAN, QU AN, NICOLE JELESOFF, AND MARTIN DYM Department of anatomy and Cell Biology, Georgetown University, School of Medicine, 3900 Reservoir Road, N.W., Washington, D.C.
ABSTRACT
A morphological and immunocytochemical study of the Golgi apparatus in pachytene spermatocytes was performed in a n effort to correlate the structure and function of this organelle during meiotic prophase. In stages 1-111 of the cycle, the Golgi complex of pachytene spermatocytes is a flattened discoid, 0.5-1 pm in diameter, composed of vesicles interspersed with classically described Golgi cisternae. During subsequent maturation of pachytene spermatocytes (stages IV-XIII), the size of the Golgi complex increases significantly, attaining a size of 2-3 pm. However, unlike pachytene spermatocytes of stages 1-111, the majority of the Golgi complex of more mature spermatocytes is characterized by a n abundance of distinct stacks of cisternae interspersed with numerous vesicles and tubules. The composition of the Golgi complex was also studied by using two monoclonal antibodies that recognize either the cis or the trans Golgi cisternae, respectively, and employing biotin-streptavidin-peroxidase immunocytochemistry in 5 pm frozen sections of testes. Immunodetection of the distinct cisternae revealed that the increase in size of the Golgi complex during maturation of pachytene spermatocytes was due predominantly to a n accumulation of trans Golgi; the amount of cis Golgi remained unchanged. The morphological data presented in this study are consistent with a n heightened secretory activity of pachytene spermatocytes during their maturation. In addition, the increase in size of the Golgi apparatus during the extensive prophase of pachytene spermatocytes may suggest that the mechanism employed by germ cells to partition the Golgi complex during the first division of meiosis varies significantly from that of somatic cells undergoing mitosis.
The Golgi apparatus of round spermatids has been plete the first division of meiosis, giving rise to secondthe subject of several rigorous investigations in the ary spermatocytes, which rapidly complete the second past (Clermont, 1956; Mollenhauer et al., 1976; Hermo meiotic division. Importantly, the second set of daughet al., 1979; 1980; Susi et al., 1980; Tang et al., 1982) ter cells is formed from a population of cells whose and these studies have been the topic of a recent review existence is quite brief. (Clermont et al., 1988). In round spermatids, the Golgi The transient event of the second meiotic division apparatus is a n elaborate organelle made up of a cortex requires that germ cells develop a n efficient and rapid (composed of distinct saccules, tubular structures, and mechanism to partition cytoplasmic organelles that exvesicles) and a medulla (consisting of a loose network of ist as a single copy in the cytoplasm. Unlike lysosomes, vesicles and tubulovesicular structures) (Clermont et for example, that exist as multiple cytoplasmic oral., 1988). With respect to its specific function, the ganelles and whose partitioning into daughter cells Golgi apparatus of round spermatids is of vital impor- may be readily accomplished by random transport, the tance in the formation of the acrosome, being involved mechanism by which the Golgi apparatus is distributed in the processing of the majority, if not all, of the en- equally to secondary spermatocytes is not known. Unzymes destined to enter the acrosome (Allison and Har- doubtedly, the mechanism of Golgi apparatus partitree, 1970; Clermont et al., 1981; Clermont and Tang, tioning in pachytene spermatocytes must be complex 1985; Clermont et al., 1988). Therefore, it is clear that and must involve a disassembly reaction a t some point the normal functioning of the Golgi apparatus is re- during meiosis. Therefore, in this study we begin the quired to ensure the correct development of spermatids during spermiogenesis. In contrast, much less is known about the Golgi apReceived January 17, 1990; accepted May 29, 1990. paratus of pachytene spermatocytes. Pachytene sperAddress to Carlos A. Suarez-Quian, Department of Anatomy and matocytes give rise to round spermatids and comprise Cell Biology, Georgetown University, School of Medicine, 3900 Resprophase, the longest phase of the first meiotic division ervoir Road, N.W., Washington, D.C. 20007. (Leblond and Clermont, 1952).At the appropriate time Present address of Dr. An Qu: Family Planning Research Institute in their development, pachytene spermatocytes com- of Sichuan, No. 15, Section 4,South People’s Road, Chengdu, China. c
1991 WILEY-LISS, INC
GOLGI COMPLEX OF I'ACHYTENE SPERMATOCYES
characterization of the Golgi apparatus of pachytene spermatocytes with respect to stages of the cycle of the seminiferous epithelium. These results suggest that the Golgi apparatus of pachytene spermatocytes behaves much differently than that of somatic cells. METHODS Light and Electron Microscopy
Six adult Sprague-Dawley rats were anesthetized with Nembutal and the testes fixed by perfusion via the abdominal aorta with 5% glutaraldehyde in 0.2 M s-Collidine buffer. After 15 mins, the testes were removed, cut into 1mm cubes, and fixed for a n additional 2 h in the same solution. The testes pieces were rinsed three times in buffer and postfixed for 1h in potassium ferrocyanide-reduced OsO, a t room temperature (Karnovsky, 1971). Next, the pieces were rinsed in buffer and dehydrated through a graded series of alcohols, followed by propylene oxide. The specimens were infiltrated with a n 1 : l mixture of propylene oxide and Epok, and embedded in Epok. For light microscopic examination, 1 pm sections were cut, mounted on slides, and stained with toluidine blue. Sections were examined with a Zeiss microscope using a 63 x Planapo, 1.4 N.A. objective. Photographs were taken with Kodak 4162 film and processed per manufacturers instructions. For electron microscopy, 60-90 nm sections were prepared using a n LKB ultramicrotome. Sections were counterstained with uranyl acetate and lead citrate and examined with a Jeol 1200 EX electron microscope. lmmunocytochemistry
Four adult Sprague-Dawley rats were killed by COz narcosis, and the testes were removed quickly and frozen in liquid nitrogen. Next, the testes were placed in a 2800 Frigocut (Reichert-Jung) Cryostat a t - 15°C and left to reach equilibrium for 2 h. Sections were cut at a thickness of 5 Fm and fixed for 10 min with either 3.7% formalin in phosphate-buffered saline, or 5 min in ice cold methanol. Histostain-SP Kits (Zymed Laboratories) for mouse primary antibodies were used exactly as described by the manufacturer's instructions to determine the precise testicular distribution of the Golgi apparatus antigens. These kits employ the biotinstreptavidin-peroxidase system to illustrate the positive, cellular immunoreaction product. The procedure is a s follows. The endogenous peroxidase activity is blocked by using a periodic acid solution (Zymed) by adding one drop to each cryostat section and incubating exactly for 45 sec. Next, endogenous avidinibiotin activity is blocked by using the AvidinlBiotin blocking kit (Zymed). First, reagent A (aviding inhibitor) is added to the sections and incubated for 10 min, followed by extensive washing with PBS. Second, reagent B (biotin inhibitor) is added, the sections are incubated for 10 min, and washed extensively with PBS. The immunocytochemical procedure involved five steps: (1 Sections were incubated in serum blocking solution for 10 min. (2) The mouse monoclonal antibodies were added and sections were incubated for 1 h a t 37°C in a moist chamber followed by extensive washes in PBS. (3)Biotinylated second antibody was added to each section and incubated for 30 min a t 37°C in a moist cham-
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ber followed by extensive washes with PBS. (4) The enzyme conjugate, streptavidin-peroxidase, was then added and sections were incubated a t room temperature for 5 min followed by extensive washing with PBS. (5) Next, the substrate-chromogen mixture, H,O,-.aminoethyl carbazole, was added to each section and incubated for 15 min a t room temperature followed by extensive washing with H,O. Control immunocytochemical studies employed included both negative (steps 1-4 below) and positive (step 5) method controls as follows: (1) Mouse monoclonal antibody was ommited; (2) hybridoma media that is negative in immunocytochemistry studies was used; (3) normal mouse sera was used; (4) hybridoma conditioned media depleted of antibodies by running through a Sepharose-protein G column (Pharmacia) was used; and (5) as a positive method control for immunocytochemistry, another monoclonal antibody was used, 37B3 (Suarez-Quian, 19881, that recognizes a nuclear lamin protein. After the immunocytochemical reaction, frozen section were counterstained with hematoxylin. To maximize visualization of the red reaction product, frozen sections were examined with a Zeiss, Planapo 6 3 ~ phase 3, 1.4 N.A. objective using a n 80A blue filter. Images were recorded on 4162 Kodak film as described above. The monoclonal antibodies 15C8 and 1 8 B l l recognize two distinct Golgi integral membrane proteins (GIMPs) and have been extensively characterized previously (Yuan et al., 1987). The 15C8 antibody recognizes a protein (GIMPc) of 130 kDa that resides in the cis and medial cisternae of the Golgi complex. In contrast, the antibody 1 8 B l l recognizes a protein (GIMPt) of 100 kDa that resides predominantly in the trans most cisternae of the Golgi complex and in the trans tubular network. In this study, the distinct 15C8 and 1 8 B l l hybridomas were maintained in culture for several days and the conditioned media harvested for use in immunocytochemistry. Approximately 1 liter from each hybridoma culture was precipitated a t a 1:l ratio with saturated ammonium sulfate a t 4"C, brought up to a 2 0 concentration ~ in PBS, and dialyzed extensively against PBS. This stock solution of monoclonal antibody was diluted 1500 and 1:lOOO and used in the immunocytochemical studies. RESULTS Light Microscopic Observations
Profiles of rat seminiferous tubules cut in cross section represent static images of different cell associations in both time and space. In the rat, the different cell associations have been extensively characterized and classified into 14 separate stages of the cycle of the seminiferous epithelium (Leblond and Clermont, 1952). Thus, by comparing the profiles of distinct cell populations comprising the 14 stages of the cycle, structural changes occurring in time, in specific cells, may be determined. With respect to the Golgi complex of pachytene spermatocytes, its morphological characterization a t the light microscopic level has heretofore not been described in the literature. As spermatocytes mature during the pachytene phase of meiosis, beginning approximately a t stage IV of the cycle, they undergo a marked increase in size.
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C.A. SUAIlEZ-QUIAN ET A L
The Golgi complex of early pachytene spermatocytes, i.e., those spermatocytes residing in the first half of the cycle, may be detected as a flattened, opaque patch in a juxtanuclear position (Fig. 1A). However, in the absence of subsequent ultrastructural characterization, i t is not clear that these profiles may be resolved a s Golgi complex. Lack of ultrastructural verification of these flattened profiles is perhaps the reason that previous miscroscopists failed to describe these areas a s forming part of the Golgi complex. In contrast, the Golgi complex of more mature spermatocytes undergoes a remarkable increase in size and thus is readily detected a t the light microscopic level (Fig. 1B). In these profiles, the Golgi complex retains its juxtanuclear position, albeit it may migrate a short distance away into the cytoplasm, and appears spherical. Within the spherical complex, opaque areas may be detected and appear arranged as thickened cisternae contained within a less dense material. Ultrastructural Observations
The identity and structural composition of apparent Golgi complexes detected in pachytene spermatocytes a t the light microscopic level are readily ascertained at the ultrastructural level (Fig. 2A-B). The flattened patch in the juxtanuclear cytoplasm, identified as Golgi complex of early pachytene spermatocytes, is composed of several flattened cisternae surrounded by a halo of vesicles and tubules (Fig. 2A). Based on its ultrastructural appearance, it is possible to assign a cis and trans side to the Golgi complex. In the example illustrated, the cis side is facing the nuclear envelope. The Golgi complex of more mature pachytene spermatocytes varies significantly from the Golgi complex of pachytene spermatocytes residing in the early part of the cycle (Fig. 2B). First, the shape of the Golgi complex is spherical. Second, a n increase in the number of vesicles, and/or tubules, associated with the Golgi complex may be observed. Third, within the Golgi complex, there is a n increase in the number of stacks of flattened cisternae, and these appear dispersed within the matrix of the Golgi apparatus in a spherical orientation. Fourth, it is not possible to assign a cis or trans side to the Golgi complex, as is the case with polarized epithelial cells, in the absence of cytochemical markers for distinct cisternae of the Golgi complex. Finally, the size of the Golgi complex increases up to 5-fold in the more mature spermatocytes. lmmunocytochemistry Results
The identity of distinct parts of the Golgi complex of early and late spermatocytes was examined in 4-5 bm frozen sections of testes prepared from adult rats by employing a biotin-streptavidin-peroxidase immunocytochemical technique. Using this technique, a positive immunoreaction is observed a t sites of deposition of the reaction product, which in this case appears reddish-brown. The sections are counterstained with hematoxylin which stain the nuclei a deep blue color. This distinction in color must be understood by the reader because the immunocytochemical data are provided in black and white. The monoclonal antibody probes used recognize integral membrane proteins of distinct cisternae of the Golgi complex: 15C8 antibody
detects a component of the cis and medial Golgi (GIMPc),whereas the 1 8 B l l antibody recognizes a constituent residing in the trans Golgi (GIMPt) and the trans-tubular network (Yuan et al., 1988). Immunocytochemistry controls used in this investigation were 2-fold (Fig. 3A-B). First, the primary monoclonal antibody was omitted andlor normal mouse sera used (Fig. 3A). In addition, condition media from nonproducing hybridomas and hybridoma conditioned media depleted of antibodies with a Sepharose-protein G column were used a s negative controls. The nuclei of cells comprising the seminiferous epithelium are readily observed because sections were counterstained with hematoxylin. Second, a positive method control was employed by using a n antibody that recognizes a component of the nuclear lamina but does not detect cytoplasmic, integral membrane proteins. In the first control study no specific immunostaining was observed, whereas in the latter case only the nucleus provided a positive immunodetection reaction (Fig. 3B). Using the 15C8 antibody that recognizes GIMPc, immunocytochemistry results revealed a random, punctate distribution of this antigen in pachytene spermatocytes (Fig. 4A-B). The immunostaining appeared to be evenly distributed in those areas of the cytoplasm that were stained positive for GIMPc; that is, no specific sites of immunoreactivity were detected. In addition, no apparent differences were detected in the distribution or appearance of GIMPc in pachytene spermatocytes associated with the different stages of the cycle of the seminiferous epithelium. The 15C8 antibody also immunodetects GIMPc in the Golgi complex of round spermatids and a prominent immunoreaction may be observed in these cells (Fig. 4, B). However, unlike the Golgi apparatus in pachytene spermatocytes, the Golgi apparatus of round spermatids, revealed by immunostaining with the 15C8 antibody, increases in size. These data are consistent with the increased size of the spermatid’s Golgi complex of round spermatids during the Golgi phase of spermiogenesis. This antibody also immunostains the Golgi apparatus of Sertoli cells and spermatogonia located a t the base of the seminiferous tubules. In contrast, the localization of GIMPt was site specific in pachytene spermatocytes, and its distribution did not coincide precisely with the disposition of GIMPc (Fig. 5A-B). The reaction product was clearly arranged in a spherical shape and was distributed in a focal manner. Further, the size of the Golgi complex, revealed by the positive immunoreaction using the 1 8 B l l antibody, increased significantly a s the pachytene spermatocytes became associated with the later stages of the cycle of the seminiferous epithelium. For example, compare the immunostaining of the Golgi complex of early and late pachytene spermatocytes illustrated in Figure 5A and B, respectively. The enlargement in the size of the Golgi complex illustrated by immunocytochemistry paralleled the growth in the dimensions of the Golgi apparatus reported in the light and ultrastructural observations of Figures 1 and 2. Specifically, the increase in the size of the spherical Golgi complex observed in pachytene spermatocytes as they progress from stages I to XIV appears to be a heterogeneous growth due solely to a n accumulation of the trans components of the Golgi cisternae. Surprisingly, unlike the
GOLGI COMPLEX OF I’ACHYTENE SI’ERMATOCYES
Fig. 1. Morphology of Golgi apparatus in pachytene spermatocytes. (A) A profile of a tubule cut in cross section a t stages 1-11 of the cycle of the seminiferous epithelium; (B) a tubule a t stages XII-XIII. Sertoli cells ( S ) ,pachytene spermatocytes (sc), and round spermatids (sp) are labeled. In A, arrows point to the Golgi apparatus that appears as an opaque patch in a perinuclear area and in B the Golgi complex is
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indicated by black-on-white circles. Note that the Golgi of the more mature pachytene spermatocytes shown in B IS significantly larger than the Golgi of the spermatocytes in A, and is characterized by an infrastructure consisting of opaque areas contained within a more translucent halo.
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C.A. SUAKEZ-QUIAN ET AL.
Fig. 2. Ultrastructure of Golgi apparatus of pachytene spermatocytes. (A) T h e Golgi complex of a stage I1 pachytene spermatocyte; (B) t h e Golgi of a stage XI11 pachytene spermatocyte. Mitochondria ( M i a r e labeled. Note the increased number of stacks of cisternae present
in the Golgi apparatus of a stage XI11 pachytene spermatocyte (compare with Fig. 1B).In this instance it is not possible to assign a cis or a trans phase to the Golgi complex. The centriole is located to t h e right side of the complex.
(;OI,GI COMPLEX O F I’ACHYTENE: SI’EIZMATOCYES
Flg. 3. Immunocytochemical controls. Frozen sections of adult r a t testes were prepared and immunostained. (A) The monoclonal antibody was omitted (similar results were also obtained using normal mouse s e r u m ) ; (B) a monoclonal antibody t h a t recognizes a lamin protein in t h e nucleus was used a s a positive method control. In B, t h e nuclei a r e immunostained hut the cytoplasm remains free of reaction product. T h e intense nuclear immunostaining observed, relative to t h e immunostaining of Figures 4 and 5, is due to the fact t h a t within a 6 y m section the reaction product is present throughout the depth of t h e section, t h a t is, I t corresponds to the nucleus whose thickness is
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greater than 6 ym. In contrast, immunostaining of t h e Golgi apparatus, detected by the presence of the reaction product, corresponds only to t h e thickness of t h a t portion of the cis or trans Golgi cisternae present within a particular section. Sections a r e counterstained with hematoxylin and thus the nuclei of elongated spermatids (arrows) appear stained. In color images they appear blue whereas t h e positive reaction product is reddish-brown. Nuclei (N)of spermatocytes a r e labeled. (See Materials and Methods for further methodological details.)
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('.A. SUAREZ-QUIAN ET AI,.
Flg. 4.Immunostaining of cis Golgi. Frozen sections of adult testes were prepared and immunostained with monoclonal antibody 15C8 that recognizes an integral membrane protein that resides in the cis most cisternae of the Golgi apparatus. (A) Stages 1-11; (B) stages VII-VIII. The brackets indicate the position of the seminiferous tubule wall. In B, the circles surround the Golgi apparatus of round spermatids which a t this stage appear enlarged. In contrast, no differences were detected in the immunostaining pattern of the cis Golgi of pachytene spermatocytes. The reaction product is observed in a
juxtanuclear distribution that corresponds with the known position of the Golgi apparatus. In color, the reddish-brown reaction product IS readily distinguished from the deep blue color of the hematoxylin counterstain of the nucleus. This antibody also immunostains the Golgi apparatus of Sertoli cells, although in these two profiles the Sertoli cell cytoplasm is not readily apparent. As before, the counterstain with hematoxylin gives rise to staining of the nuclei of elongated spermatids and spermatocytes tN).
GOLGI COMPLEX OF I’ACHYTENE SPERMATOCYES
Fig. 5. Immunostaining of trans Golgi. Frozen sections of adult testes were prepared and the trans most Golgi cisternae were immunostained with monoclonal antibody 1 8 B l l . The wails of t h e seminiferous tubules, cut in cross section, a r e indicated by t h e brackets and t h e nuclei ( N ! of pachytene spermatocytes a r e labeled. (A,B) The trans Golgi of round spermatids is indicated by circles and arrows point to
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t h e trans Golgi of pachytene spermatocytes. In A a n early stage (11III! seminiferous epithelium is illustrated, whereas a late stage (XIIXIII) seminiferous epithelium is shown in B. Note t h e significant difference in size of the trans Golgi of pachytene spermatocytes residing in a n early stage versus a late stage seminiferous epithelium.
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C . A . SUAREZ-QUIAN ET AL.
15C8 antibody, the 1 8 B l l antibody does not appear to immunostain the Golgi complex of the Sertoli cell. DISCUSSION
The Golgi apparatus was examined a t the light and electron microscopic levels to determine its structure in pachytene spermatocytes. In addition, the intracellular distribution of distinct regions of the Golgi apparatus, the trans and cis saccules, was determined by immunocytochemical methodology. Our results demonstrate that the Golgi apparatus may be detected in pachytene spermatocytes in early stages of the cycle of the seminiferous epithelium, a t the level of resolution provided by the light microscope, and that a s the pachytene spermatocytes undergo maturation the Golgi apparatus increases significantly in size. Ultrastructural observations revealed that the composition of the Golgi apparatus varies between early and late pachytene spermatocytes. (1) The Golgi apparatus of early pachytene spermatocytes is characterized by a stack of classically arranged cisternae surrounded by tubulovesicular structures and vesicles. (2) In contrast, the Golgi apparatus of late pachytene spermatocytes is composed of distinct Golgi complexes contained within one large juxtanuclear Golgi area and exhibits large numbers of vesicles and/or tubulovesicular structures. Further analysis a t the ultrastructural level will reveal whether the increase in the apparent number of Golgi cisternae is a quantitative phenomena, or whether the increase represents hypertrophy of the cisternae present in the early pachytene spermatocytes. The differential ultrastructural observations made between early and late pachytene spermatocyte Golgi apparatus are consistent with immunocytochemical results reported in the present study. Immunostaining demonstrated that the increase in size of the Golgi apparatus observed during maturation of pachytene spermatocytes appears to be due predominantly to the trans most elements of the Golgi cisternae. In addition, it is likely that some of the increase in size is due to immunostaining of the trans tubulovesicular structurers andlor vesicles, since in the original characterization of this antibody, coated vesicles of the trans-tubular network were clearly immunostained (Yuan et al., 1987). No change was observed in the immunostaining pattern of the cis most Golgi cisternae in pachytene spermatocytes residing at different stages of the cycle. However, unlike in the initial report characterizing the 15C8 antibody a s a specific marker of the cis and medial Golgi cisternae, the immunostaining pattern observed in the present study is remeniscent of small vesicle immunostaining. These observations may suggest that transitional vesicles between the endoplasmic reticulum and the cis Golgi may also contain the specific protein recognized by the 15C8 monoclonal antibody. Clearly, these observations require additional ultrastructural immunocytochemical studies to verify precisely the identity of the structures immunostained with this antibody. Organelle distribution during the process of cell division must be under precise regulation to ensure that the resulting two daughter cells receive a n equal complement of each organelle. In the case of multiple copy organelles, e.g., mitochondria, a means by which cells may accomplish this distribution is to partition the or-
ganelles equally via random transport during telophase (Birky, 1983). In contrast, the Golgi apparatus is a single copy organelle (Rambourg et al., 1974, 1979, 1981) and cells must accomplish equal partitioning by a n as yet unknown mechanism, albeit one that must undoubtedly be under exact and likely complex control. For example, previous data (Robbins and Gonatas, 1964; Maul and Brinkly, 1970; Zeligs and Wollman, 1979; Hiller and Weber, 1982) and more recent evidence (Lucocq e t al., 1987) obtained in HeLa cells suggest that the Golgi apparatus undergoes a disassembly reaction prior to the onset of mitosis, resulting in the formation of small Golgi tubulovesicular clusters and free vesicles. The Golgi clusters and/or free vesicles have been proposed to serve in the partitioning of the Golgi membranes a t telophase, since they amplify the single copy Golgi complex into a multiple copy cytoplasmic organelle (Lucocq and Warren, 1987). Subsequently, the Golgi clusters appear to reassemble by a process termed “accretion of the Golgi clusters,” likely involving several intermediate steps not yet characterized fully (Lucocq et al., 1989). In addition, the endoplasmic reticulum has been implicated in the contribution of membranes to the Golgi apparatus a t telophase (Featherstone et al., 1985). Therefore, a means by which germ cells may process cytoplasmic organelles, e.g., the Golgi complex, during spermatogenesis is to recruit a mechanism similar to that evolved by somatic cells. However, our results suggest that the mechanism employed by germ cells to achieve Golgi apparatus partitioning may differ somewhat from that observed in somatic cells. First, the pachytene spermatocytes represent the longest phase of prophase of the first division of meiosis and may be observed during the first 12 stages of the cycle of the seminiferous epithelium (Leblond and Clermont, 1952). At stage 13, the pachytene spermatocytes become known a s diplotene spermatocytes and quickly terminate anaphase I and telophase I giving rise to two secondary spermatocytes. The latter exist for only a few hours and then undergo the second division of meiosis, giving rise to four round spermatids (Perey et al., 1961). Although the two meiotic divisions in themselves do not represent significant differences in the overall theme of somatic cell partitioning of organelles, it must be emphasized that the two divisions occur during a brief period of time, a t most 8 h (Perey et al., 1961). Thus, i t seems unlikely that during their short life span secondary spermatocytes are capable of synthesizing required proteins for the completion of the second division of meiosis and generate sufficient organelle-specific proteins to be subsequently partitioned into two round spermatids. Moreover, a model of somatic cell Golgi complex disassembly would suggest that intermediate size Golgi clusters should be readily detected during the second division of meiosis a s a function of time. Immunostaining of the Golgi apparatus a t this time, presumably undergoing a disassembly reaction, should give rise to a gradient in the size of the Golgi complex a s revealed by immunocytochemistry. However, the immunostaining of the Golgi apparatus of late stage spermatocytes or secondary spermatocytes revealed by the antiGIMPt antibody was not consistent with the predicted diminution in size of Golgi clusters and/or tubulovesic-
GOLGI COMPLEX OF PACHYTENE SPERMATOCYES
ular structures. Nevertheless, a rigorous ultrastructural study of changes incurred by secondary spermatocyte’s Golgi apparatus during meoisis I1 must be performed to verify our qualitative immunocytochemical results. Second, unlike somatic cells, as pachytene spermatocytes approach the first division of meiosis their Golgi apparatus increases significantly in size (Figs. 1and 2). In addition, the composition of the Golgi apparatus appears altered; the Golgi apparatus of late pachytene spermatocytes appears to contain multiple stacks of Golgi cisternae (Fig. 2). This is in marked contrast to the ultrastructural appearance of the Golgi apparatus of somatic cells that is beginning to undergo disassembly. Third, strong evidence supports a n involvement of microtubules in the process of partitioning of the small Golgi cluster once these have undergone a disassembly reaction (Allan and Kreiss, 1986). Thus, we expected to find microtubules associated with the Golgi apparatus of pachytene spermatocytes at the time of the Golgi complex Partitioning. However, unlike the Golgi apparatus in somatic cells, in our study only large Golgi apparatuses, containing several stacks of Golgi cisternae, were qualitatively observed to be associated with centrioles (Fig. a), microtubule organizing centers. These results are consistent with the recruitment of microtubules by the large Golgi apparatuses a t the time that initiation of the first division of meiosis occurs. Importantly, the results provide indirect evidence that the large Golgi apparatus may begin the process of partitioning without first undergoing a disassembly reaction. Therefore our results suggest that partitioning of the spermatocyte’s Golgi apparatus during the first meiotic division is unlikely to involve a disassembly reaction terminating in small Golgi clusters. Instead, we favor a mechanism whereby the Golgi apparatus of late pachytene spermatocytes generates several stacks of Golgi cisternae and these undergo a partitioning into the subsequent secondary spermatocytes. Whether the second meiotic division involves a dissolution of the Golgi apparatus, however, remains to be determined. Although disassembly of the Golgi apparatus into small vesicle clusters may occur during the second meiotic division, additional studies, in particular a n ultrastructural examination of secondary spermatocytes, will be required to resolve this issue. Nevertheless, because of the specific needs of the round spermatids that result from the second meiotic division it is possible to speculate that partitioning of the Golgi complex a s discrete units may accommodate the onset of spermiogenesis. Round spermatids a t stage one of the cycle enter the Golgi phase of spermiogenesis which will eventually lead to the formation of the acrosome (Clermont et al., 1988).Possibly, round spermatids minimize energy expenditures by eliminating the assembly reaction of Golgi clusters into mature Golgi apparatuses. In this regard i t must be emphasized that the generation of the acrosome is not a trivial phenomena, since the acrosome of rat spermatids eventually occupies a major portion of the cell volume. Additionally, it is not yet clear whether the Golgi apparatus is required during meiosis. For example, a structured Golgi complex present during meiosis may permit the processing of
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proteins during cell division andlor leave the newly formed round spermatids primed to begin formation of the acrosome. However, we wish to emphasize that our interpretation of the mechanism by which pachytene spermatocytes partition the Golgi apparatus is preliminary at this time and awaits verification by ultrastructural verification. Moreover, the recent studies demonstrating a role of pachytene spermatocyte products in modulating Sertoli cell function may now be understood within the context of the present data (Djakiew and Dym, 1989; Castellon et al., 1989). Increase in the size of the Golgi apparatus during pachytene spermatocyte maturation, in particular the trans most cisternae, is consistent with this observation and may provide the structural basis for the apparent increase in the secretory activity of pachytene spermatocytes. ACKNOWLEDGMENTS
We wish to acknowledge the most helpful comments of the reviewers of this manuscript. This work was supported by NIH Grants HD23484 to C.A.S.-Q. and HD16260 to M.D. LITERATURE CITED Allan, V.J., and T.E. Kreis 1986 A microtubule-binding protein associated with membranes of the Golgi apparatus. J. Cell. Biol., 2229-2239. Allison, A.C., and E.F. Hartree 1970 Lysosomal enzymes in the acrosome and their possible role in fertilization. J. Reprod. Fertil., 21.501-515. Birky, C.W. 1983 The partitioning of cytoplasmic organelles a t cell division. Int. Rev. Cytol., l5:49-89. Clermont, Y. 1956 The Golgi zone of the r a t spermatid and its role in the formation of cytoplasmic vesicles. J. Biophys. Biochem. Cyt. Suppl., 2t119-122. Clermont, Y., and X.M. Tang 1985 Glycoprotein synthesis in the Golgi apparatus of spermatids during spermiogenesis of the rat. Anat. Rec., 213.33-43. Clermont, Y., M. Lalli, and A. Rambourg 1981 Ultrastructural localization of nicotinamide adenine dinucleotide phosphatase (NADPase), thiamine pyrophosphatase (TPPase), and cytidine monophosphatase (CMPase)in the Golgi apparatus of early spermatids of the rat. Anat. Rec., 201.613-622. Clermont, Y., L. Hermo, A. Rambourg, and G. Thorne-Tjomsland 1988 Structural and cytochemical characteristics of the Golgi apparatus during the formation of the acrosomic system in r a t spermatids. In: Golgi, Lysosomes and Centriole Events in Early Spermiogenesis: Targets for Male Fertility Regulation. D. Hamilton and G.M.H. Waites, eds. Cambridge University Press, London. Djakiew, D. and M. Dym 1988 Pachytene spermatocyte proteins influence Sertoli cell function. Biol. Reprod., 39t1193-1205. Featherstone, C., G. Griffth, and G. Warren 1985 Newly synthesized G protein of vesicular stomatitis virus is not transported to the Golgi complex in mitotic cells. J. Cell Biol., 101.2036-2046. Hermo, L., Y. Clermont, and A. Rambourg 1979 Endoplasmic reticulum-Golgi apparatus relationships in the rat spermatid. Anat. Rec., 193.243-256. Hermo, L., A. Rambourg, and Y. Clermont 1980 Three-dimensional architecture of the cortical region of the Golgi apparatus in rat spermatids. Am. J . Anat., 157t357-373. Hiller, G., and K. Weber 1982 Golgi detection in mitotic and interphase cell by antibodies to secreted galactosyltransferase. Exp. Cell Res., 142t85-94. Karnovsky, M.J. 1971 Use of ferrocyanide-reduced osmium tetroxide in electron microscopy. J. Cell Biol., Abstract 284. Leblond, C.P., and Y. Clermont 1952 Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann. N.Y. Acad. Sci., 5Fit548-573 Lucocq, J.M., and G Warren 1987 Fragmentation and partitioning of the Golgi apparatus in HeLa cells. EMBO J., 6t3239-3246. Lucocq, J.M., J. Pryde, E. Berger, and G. Warren 1987 A mitotic form of the Golgi apparatus in HeLa cells. J. Cell Biol., 104.865-874. Lucocq, J.M., E.G. Berger, and G. Warren 1989 Mitotic Golgi frag-
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ments in HeLa cells and their role in the reassembly pathway. J. Cell Biol., 463-474. Maul, G.G., and B.R. Brinkley 1970 The Golgi apparatus during mitosis in human melanoma cells in uitro. Cancer Res., 30r23262335. Mollenhauer, H., B.S. Hass, and J. Morre 1976 Membranous transformations in Golgi apparatus of rat spermatids. A role for thick cisternae and two classes of coated vesicles in acrosome formation. J. Microsc. Biol. Cell., 27.33-36. Perey, B., Y. Clermont, and C.P. Leblond 1961 The wave of the seminiferous epithelium in the rat. Am. J. Anat., 108:47-77. Bambourg, A,, Y. Clermont, and A. Marraud 1974 Three-dimensional structure of the osmium-impregnated Golgi apparatus as seen in the high voltage electron microscope. Am. J. Anat., 140.27-46. Rambourg, A., Y. Clermont, and L. Hermo Three-dimensional architecture of the Golgi apparatus in Sertoli cells of the rat. Am. J. Anat., 154.455-476. Rambourg, A., Y. Clermont, and L. Hermo 1981 Three-dimensional structure of the Golgi apparatus. Methods Cell Biol., 23.155-166. ~~
Robbins, E., and N.K. Gonatas 1964 The ultrastructure of a mammalian cell during the mitotic cycle. J. Cell Biol., 21:429-463. Suarez-Quian, C.A. 1988 Differential cell surface expression of four lysosomal integral membrane proteins (LIMPS) in normal rat kidney cells. Tissue Cell, 20.35-46. 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. Tang, X.M., M.F. Lalli, and Y. Clermont 1982 A cytochemical study fo the Golgi apparatus of the spermatid during spermiogenesis in the rat. Am. J. Anat., 163:283-293. Yuan, L., J.G. Barriocanal, J.S. Bonifacino, and I.V. Sandoval 1987 Two integral membrane proteins located in the cis-middle and trans-part of the Golgi system acquired sialated N-linked carbohydrates and display different turnovers and sensitivity to CAMP-dependent phosphorylation. J. Cell Biol., 105t215-227. Zeligs, J.D., and S.H. Wollman 1979 Mitosis in rat thyroid epithelial cells in vivo. I. Ultrastructural changes in cytoplasmic organelles during the mitotic cycle. J. Ultrastruct. Res., 66:53-77.
The Golgi Apparatus of Rat Pachytene Spermatocytes During Spermatogenesis CARLOS A. SUAREZ-QUIAN, QU AN, NICOLE JELESOFF, AND MARTIN DYM Department of anatomy and Cell Biology, Georgetown University, School of Medicine, 3900 Reservoir Road, N.W., Washington, D.C.
ABSTRACT
A morphological and immunocytochemical study of the Golgi apparatus in pachytene spermatocytes was performed in a n effort to correlate the structure and function of this organelle during meiotic prophase. In stages 1-111 of the cycle, the Golgi complex of pachytene spermatocytes is a flattened discoid, 0.5-1 pm in diameter, composed of vesicles interspersed with classically described Golgi cisternae. During subsequent maturation of pachytene spermatocytes (stages IV-XIII), the size of the Golgi complex increases significantly, attaining a size of 2-3 pm. However, unlike pachytene spermatocytes of stages 1-111, the majority of the Golgi complex of more mature spermatocytes is characterized by a n abundance of distinct stacks of cisternae interspersed with numerous vesicles and tubules. The composition of the Golgi complex was also studied by using two monoclonal antibodies that recognize either the cis or the trans Golgi cisternae, respectively, and employing biotin-streptavidin-peroxidase immunocytochemistry in 5 pm frozen sections of testes. Immunodetection of the distinct cisternae revealed that the increase in size of the Golgi complex during maturation of pachytene spermatocytes was due predominantly to a n accumulation of trans Golgi; the amount of cis Golgi remained unchanged. The morphological data presented in this study are consistent with a n heightened secretory activity of pachytene spermatocytes during their maturation. In addition, the increase in size of the Golgi apparatus during the extensive prophase of pachytene spermatocytes may suggest that the mechanism employed by germ cells to partition the Golgi complex during the first division of meiosis varies significantly from that of somatic cells undergoing mitosis.
The Golgi apparatus of round spermatids has been plete the first division of meiosis, giving rise to secondthe subject of several rigorous investigations in the ary spermatocytes, which rapidly complete the second past (Clermont, 1956; Mollenhauer et al., 1976; Hermo meiotic division. Importantly, the second set of daughet al., 1979; 1980; Susi et al., 1980; Tang et al., 1982) ter cells is formed from a population of cells whose and these studies have been the topic of a recent review existence is quite brief. (Clermont et al., 1988). In round spermatids, the Golgi The transient event of the second meiotic division apparatus is a n elaborate organelle made up of a cortex requires that germ cells develop a n efficient and rapid (composed of distinct saccules, tubular structures, and mechanism to partition cytoplasmic organelles that exvesicles) and a medulla (consisting of a loose network of ist as a single copy in the cytoplasm. Unlike lysosomes, vesicles and tubulovesicular structures) (Clermont et for example, that exist as multiple cytoplasmic oral., 1988). With respect to its specific function, the ganelles and whose partitioning into daughter cells Golgi apparatus of round spermatids is of vital impor- may be readily accomplished by random transport, the tance in the formation of the acrosome, being involved mechanism by which the Golgi apparatus is distributed in the processing of the majority, if not all, of the en- equally to secondary spermatocytes is not known. Unzymes destined to enter the acrosome (Allison and Har- doubtedly, the mechanism of Golgi apparatus partitree, 1970; Clermont et al., 1981; Clermont and Tang, tioning in pachytene spermatocytes must be complex 1985; Clermont et al., 1988). Therefore, it is clear that and must involve a disassembly reaction a t some point the normal functioning of the Golgi apparatus is re- during meiosis. Therefore, in this study we begin the quired to ensure the correct development of spermatids during spermiogenesis. In contrast, much less is known about the Golgi apReceived January 17, 1990; accepted May 29, 1990. paratus of pachytene spermatocytes. Pachytene sperAddress to Carlos A. Suarez-Quian, Department of Anatomy and matocytes give rise to round spermatids and comprise Cell Biology, Georgetown University, School of Medicine, 3900 Resprophase, the longest phase of the first meiotic division ervoir Road, N.W., Washington, D.C. 20007. (Leblond and Clermont, 1952).At the appropriate time Present address of Dr. An Qu: Family Planning Research Institute in their development, pachytene spermatocytes com- of Sichuan, No. 15, Section 4,South People’s Road, Chengdu, China. c
1991 WILEY-LISS, INC
GOLGI COMPLEX OF I'ACHYTENE SPERMATOCYES
characterization of the Golgi apparatus of pachytene spermatocytes with respect to stages of the cycle of the seminiferous epithelium. These results suggest that the Golgi apparatus of pachytene spermatocytes behaves much differently than that of somatic cells. METHODS Light and Electron Microscopy
Six adult Sprague-Dawley rats were anesthetized with Nembutal and the testes fixed by perfusion via the abdominal aorta with 5% glutaraldehyde in 0.2 M s-Collidine buffer. After 15 mins, the testes were removed, cut into 1mm cubes, and fixed for a n additional 2 h in the same solution. The testes pieces were rinsed three times in buffer and postfixed for 1h in potassium ferrocyanide-reduced OsO, a t room temperature (Karnovsky, 1971). Next, the pieces were rinsed in buffer and dehydrated through a graded series of alcohols, followed by propylene oxide. The specimens were infiltrated with a n 1 : l mixture of propylene oxide and Epok, and embedded in Epok. For light microscopic examination, 1 pm sections were cut, mounted on slides, and stained with toluidine blue. Sections were examined with a Zeiss microscope using a 63 x Planapo, 1.4 N.A. objective. Photographs were taken with Kodak 4162 film and processed per manufacturers instructions. For electron microscopy, 60-90 nm sections were prepared using a n LKB ultramicrotome. Sections were counterstained with uranyl acetate and lead citrate and examined with a Jeol 1200 EX electron microscope. lmmunocytochemistry
Four adult Sprague-Dawley rats were killed by COz narcosis, and the testes were removed quickly and frozen in liquid nitrogen. Next, the testes were placed in a 2800 Frigocut (Reichert-Jung) Cryostat a t - 15°C and left to reach equilibrium for 2 h. Sections were cut at a thickness of 5 Fm and fixed for 10 min with either 3.7% formalin in phosphate-buffered saline, or 5 min in ice cold methanol. Histostain-SP Kits (Zymed Laboratories) for mouse primary antibodies were used exactly as described by the manufacturer's instructions to determine the precise testicular distribution of the Golgi apparatus antigens. These kits employ the biotinstreptavidin-peroxidase system to illustrate the positive, cellular immunoreaction product. The procedure is a s follows. The endogenous peroxidase activity is blocked by using a periodic acid solution (Zymed) by adding one drop to each cryostat section and incubating exactly for 45 sec. Next, endogenous avidinibiotin activity is blocked by using the AvidinlBiotin blocking kit (Zymed). First, reagent A (aviding inhibitor) is added to the sections and incubated for 10 min, followed by extensive washing with PBS. Second, reagent B (biotin inhibitor) is added, the sections are incubated for 10 min, and washed extensively with PBS. The immunocytochemical procedure involved five steps: (1 Sections were incubated in serum blocking solution for 10 min. (2) The mouse monoclonal antibodies were added and sections were incubated for 1 h a t 37°C in a moist chamber followed by extensive washes in PBS. (3)Biotinylated second antibody was added to each section and incubated for 30 min a t 37°C in a moist cham-
17
ber followed by extensive washes with PBS. (4) The enzyme conjugate, streptavidin-peroxidase, was then added and sections were incubated a t room temperature for 5 min followed by extensive washing with PBS. (5) Next, the substrate-chromogen mixture, H,O,-.aminoethyl carbazole, was added to each section and incubated for 15 min a t room temperature followed by extensive washing with H,O. Control immunocytochemical studies employed included both negative (steps 1-4 below) and positive (step 5) method controls as follows: (1) Mouse monoclonal antibody was ommited; (2) hybridoma media that is negative in immunocytochemistry studies was used; (3) normal mouse sera was used; (4) hybridoma conditioned media depleted of antibodies by running through a Sepharose-protein G column (Pharmacia) was used; and (5) as a positive method control for immunocytochemistry, another monoclonal antibody was used, 37B3 (Suarez-Quian, 19881, that recognizes a nuclear lamin protein. After the immunocytochemical reaction, frozen section were counterstained with hematoxylin. To maximize visualization of the red reaction product, frozen sections were examined with a Zeiss, Planapo 6 3 ~ phase 3, 1.4 N.A. objective using a n 80A blue filter. Images were recorded on 4162 Kodak film as described above. The monoclonal antibodies 15C8 and 1 8 B l l recognize two distinct Golgi integral membrane proteins (GIMPs) and have been extensively characterized previously (Yuan et al., 1987). The 15C8 antibody recognizes a protein (GIMPc) of 130 kDa that resides in the cis and medial cisternae of the Golgi complex. In contrast, the antibody 1 8 B l l recognizes a protein (GIMPt) of 100 kDa that resides predominantly in the trans most cisternae of the Golgi complex and in the trans tubular network. In this study, the distinct 15C8 and 1 8 B l l hybridomas were maintained in culture for several days and the conditioned media harvested for use in immunocytochemistry. Approximately 1 liter from each hybridoma culture was precipitated a t a 1:l ratio with saturated ammonium sulfate a t 4"C, brought up to a 2 0 concentration ~ in PBS, and dialyzed extensively against PBS. This stock solution of monoclonal antibody was diluted 1500 and 1:lOOO and used in the immunocytochemical studies. RESULTS Light Microscopic Observations
Profiles of rat seminiferous tubules cut in cross section represent static images of different cell associations in both time and space. In the rat, the different cell associations have been extensively characterized and classified into 14 separate stages of the cycle of the seminiferous epithelium (Leblond and Clermont, 1952). Thus, by comparing the profiles of distinct cell populations comprising the 14 stages of the cycle, structural changes occurring in time, in specific cells, may be determined. With respect to the Golgi complex of pachytene spermatocytes, its morphological characterization a t the light microscopic level has heretofore not been described in the literature. As spermatocytes mature during the pachytene phase of meiosis, beginning approximately a t stage IV of the cycle, they undergo a marked increase in size.
18
C.A. SUAIlEZ-QUIAN ET A L
The Golgi complex of early pachytene spermatocytes, i.e., those spermatocytes residing in the first half of the cycle, may be detected as a flattened, opaque patch in a juxtanuclear position (Fig. 1A). However, in the absence of subsequent ultrastructural characterization, i t is not clear that these profiles may be resolved a s Golgi complex. Lack of ultrastructural verification of these flattened profiles is perhaps the reason that previous miscroscopists failed to describe these areas a s forming part of the Golgi complex. In contrast, the Golgi complex of more mature spermatocytes undergoes a remarkable increase in size and thus is readily detected a t the light microscopic level (Fig. 1B). In these profiles, the Golgi complex retains its juxtanuclear position, albeit it may migrate a short distance away into the cytoplasm, and appears spherical. Within the spherical complex, opaque areas may be detected and appear arranged as thickened cisternae contained within a less dense material. Ultrastructural Observations
The identity and structural composition of apparent Golgi complexes detected in pachytene spermatocytes a t the light microscopic level are readily ascertained at the ultrastructural level (Fig. 2A-B). The flattened patch in the juxtanuclear cytoplasm, identified as Golgi complex of early pachytene spermatocytes, is composed of several flattened cisternae surrounded by a halo of vesicles and tubules (Fig. 2A). Based on its ultrastructural appearance, it is possible to assign a cis and trans side to the Golgi complex. In the example illustrated, the cis side is facing the nuclear envelope. The Golgi complex of more mature pachytene spermatocytes varies significantly from the Golgi complex of pachytene spermatocytes residing in the early part of the cycle (Fig. 2B). First, the shape of the Golgi complex is spherical. Second, a n increase in the number of vesicles, and/or tubules, associated with the Golgi complex may be observed. Third, within the Golgi complex, there is a n increase in the number of stacks of flattened cisternae, and these appear dispersed within the matrix of the Golgi apparatus in a spherical orientation. Fourth, it is not possible to assign a cis or trans side to the Golgi complex, as is the case with polarized epithelial cells, in the absence of cytochemical markers for distinct cisternae of the Golgi complex. Finally, the size of the Golgi complex increases up to 5-fold in the more mature spermatocytes. lmmunocytochemistry Results
The identity of distinct parts of the Golgi complex of early and late spermatocytes was examined in 4-5 bm frozen sections of testes prepared from adult rats by employing a biotin-streptavidin-peroxidase immunocytochemical technique. Using this technique, a positive immunoreaction is observed a t sites of deposition of the reaction product, which in this case appears reddish-brown. The sections are counterstained with hematoxylin which stain the nuclei a deep blue color. This distinction in color must be understood by the reader because the immunocytochemical data are provided in black and white. The monoclonal antibody probes used recognize integral membrane proteins of distinct cisternae of the Golgi complex: 15C8 antibody
detects a component of the cis and medial Golgi (GIMPc),whereas the 1 8 B l l antibody recognizes a constituent residing in the trans Golgi (GIMPt) and the trans-tubular network (Yuan et al., 1988). Immunocytochemistry controls used in this investigation were 2-fold (Fig. 3A-B). First, the primary monoclonal antibody was omitted andlor normal mouse sera used (Fig. 3A). In addition, condition media from nonproducing hybridomas and hybridoma conditioned media depleted of antibodies with a Sepharose-protein G column were used a s negative controls. The nuclei of cells comprising the seminiferous epithelium are readily observed because sections were counterstained with hematoxylin. Second, a positive method control was employed by using a n antibody that recognizes a component of the nuclear lamina but does not detect cytoplasmic, integral membrane proteins. In the first control study no specific immunostaining was observed, whereas in the latter case only the nucleus provided a positive immunodetection reaction (Fig. 3B). Using the 15C8 antibody that recognizes GIMPc, immunocytochemistry results revealed a random, punctate distribution of this antigen in pachytene spermatocytes (Fig. 4A-B). The immunostaining appeared to be evenly distributed in those areas of the cytoplasm that were stained positive for GIMPc; that is, no specific sites of immunoreactivity were detected. In addition, no apparent differences were detected in the distribution or appearance of GIMPc in pachytene spermatocytes associated with the different stages of the cycle of the seminiferous epithelium. The 15C8 antibody also immunodetects GIMPc in the Golgi complex of round spermatids and a prominent immunoreaction may be observed in these cells (Fig. 4, B). However, unlike the Golgi apparatus in pachytene spermatocytes, the Golgi apparatus of round spermatids, revealed by immunostaining with the 15C8 antibody, increases in size. These data are consistent with the increased size of the spermatid’s Golgi complex of round spermatids during the Golgi phase of spermiogenesis. This antibody also immunostains the Golgi apparatus of Sertoli cells and spermatogonia located a t the base of the seminiferous tubules. In contrast, the localization of GIMPt was site specific in pachytene spermatocytes, and its distribution did not coincide precisely with the disposition of GIMPc (Fig. 5A-B). The reaction product was clearly arranged in a spherical shape and was distributed in a focal manner. Further, the size of the Golgi complex, revealed by the positive immunoreaction using the 1 8 B l l antibody, increased significantly a s the pachytene spermatocytes became associated with the later stages of the cycle of the seminiferous epithelium. For example, compare the immunostaining of the Golgi complex of early and late pachytene spermatocytes illustrated in Figure 5A and B, respectively. The enlargement in the size of the Golgi complex illustrated by immunocytochemistry paralleled the growth in the dimensions of the Golgi apparatus reported in the light and ultrastructural observations of Figures 1 and 2. Specifically, the increase in the size of the spherical Golgi complex observed in pachytene spermatocytes as they progress from stages I to XIV appears to be a heterogeneous growth due solely to a n accumulation of the trans components of the Golgi cisternae. Surprisingly, unlike the
GOLGI COMPLEX OF I’ACHYTENE SI’ERMATOCYES
Fig. 1. Morphology of Golgi apparatus in pachytene spermatocytes. (A) A profile of a tubule cut in cross section a t stages 1-11 of the cycle of the seminiferous epithelium; (B) a tubule a t stages XII-XIII. Sertoli cells ( S ) ,pachytene spermatocytes (sc), and round spermatids (sp) are labeled. In A, arrows point to the Golgi apparatus that appears as an opaque patch in a perinuclear area and in B the Golgi complex is
19
indicated by black-on-white circles. Note that the Golgi of the more mature pachytene spermatocytes shown in B IS significantly larger than the Golgi of the spermatocytes in A, and is characterized by an infrastructure consisting of opaque areas contained within a more translucent halo.
20
C.A. SUAKEZ-QUIAN ET AL.
Fig. 2. Ultrastructure of Golgi apparatus of pachytene spermatocytes. (A) T h e Golgi complex of a stage I1 pachytene spermatocyte; (B) t h e Golgi of a stage XI11 pachytene spermatocyte. Mitochondria ( M i a r e labeled. Note the increased number of stacks of cisternae present
in the Golgi apparatus of a stage XI11 pachytene spermatocyte (compare with Fig. 1B).In this instance it is not possible to assign a cis or a trans phase to the Golgi complex. The centriole is located to t h e right side of the complex.
(;OI,GI COMPLEX O F I’ACHYTENE: SI’EIZMATOCYES
Flg. 3. Immunocytochemical controls. Frozen sections of adult r a t testes were prepared and immunostained. (A) The monoclonal antibody was omitted (similar results were also obtained using normal mouse s e r u m ) ; (B) a monoclonal antibody t h a t recognizes a lamin protein in t h e nucleus was used a s a positive method control. In B, t h e nuclei a r e immunostained hut the cytoplasm remains free of reaction product. T h e intense nuclear immunostaining observed, relative to t h e immunostaining of Figures 4 and 5, is due to the fact t h a t within a 6 y m section the reaction product is present throughout the depth of t h e section, t h a t is, I t corresponds to the nucleus whose thickness is
21
greater than 6 ym. In contrast, immunostaining of t h e Golgi apparatus, detected by the presence of the reaction product, corresponds only to t h e thickness of t h a t portion of the cis or trans Golgi cisternae present within a particular section. Sections a r e counterstained with hematoxylin and thus the nuclei of elongated spermatids (arrows) appear stained. In color images they appear blue whereas t h e positive reaction product is reddish-brown. Nuclei (N)of spermatocytes a r e labeled. (See Materials and Methods for further methodological details.)
22
('.A. SUAREZ-QUIAN ET AI,.
Flg. 4.Immunostaining of cis Golgi. Frozen sections of adult testes were prepared and immunostained with monoclonal antibody 15C8 that recognizes an integral membrane protein that resides in the cis most cisternae of the Golgi apparatus. (A) Stages 1-11; (B) stages VII-VIII. The brackets indicate the position of the seminiferous tubule wall. In B, the circles surround the Golgi apparatus of round spermatids which a t this stage appear enlarged. In contrast, no differences were detected in the immunostaining pattern of the cis Golgi of pachytene spermatocytes. The reaction product is observed in a
juxtanuclear distribution that corresponds with the known position of the Golgi apparatus. In color, the reddish-brown reaction product IS readily distinguished from the deep blue color of the hematoxylin counterstain of the nucleus. This antibody also immunostains the Golgi apparatus of Sertoli cells, although in these two profiles the Sertoli cell cytoplasm is not readily apparent. As before, the counterstain with hematoxylin gives rise to staining of the nuclei of elongated spermatids and spermatocytes tN).
GOLGI COMPLEX OF I’ACHYTENE SPERMATOCYES
Fig. 5. Immunostaining of trans Golgi. Frozen sections of adult testes were prepared and the trans most Golgi cisternae were immunostained with monoclonal antibody 1 8 B l l . The wails of t h e seminiferous tubules, cut in cross section, a r e indicated by t h e brackets and t h e nuclei ( N ! of pachytene spermatocytes a r e labeled. (A,B) The trans Golgi of round spermatids is indicated by circles and arrows point to
23
t h e trans Golgi of pachytene spermatocytes. In A a n early stage (11III! seminiferous epithelium is illustrated, whereas a late stage (XIIXIII) seminiferous epithelium is shown in B. Note t h e significant difference in size of the trans Golgi of pachytene spermatocytes residing in a n early stage versus a late stage seminiferous epithelium.
24
C . A . SUAREZ-QUIAN ET AL.
15C8 antibody, the 1 8 B l l antibody does not appear to immunostain the Golgi complex of the Sertoli cell. DISCUSSION
The Golgi apparatus was examined a t the light and electron microscopic levels to determine its structure in pachytene spermatocytes. In addition, the intracellular distribution of distinct regions of the Golgi apparatus, the trans and cis saccules, was determined by immunocytochemical methodology. Our results demonstrate that the Golgi apparatus may be detected in pachytene spermatocytes in early stages of the cycle of the seminiferous epithelium, a t the level of resolution provided by the light microscope, and that a s the pachytene spermatocytes undergo maturation the Golgi apparatus increases significantly in size. Ultrastructural observations revealed that the composition of the Golgi apparatus varies between early and late pachytene spermatocytes. (1) The Golgi apparatus of early pachytene spermatocytes is characterized by a stack of classically arranged cisternae surrounded by tubulovesicular structures and vesicles. (2) In contrast, the Golgi apparatus of late pachytene spermatocytes is composed of distinct Golgi complexes contained within one large juxtanuclear Golgi area and exhibits large numbers of vesicles and/or tubulovesicular structures. Further analysis a t the ultrastructural level will reveal whether the increase in the apparent number of Golgi cisternae is a quantitative phenomena, or whether the increase represents hypertrophy of the cisternae present in the early pachytene spermatocytes. The differential ultrastructural observations made between early and late pachytene spermatocyte Golgi apparatus are consistent with immunocytochemical results reported in the present study. Immunostaining demonstrated that the increase in size of the Golgi apparatus observed during maturation of pachytene spermatocytes appears to be due predominantly to the trans most elements of the Golgi cisternae. In addition, it is likely that some of the increase in size is due to immunostaining of the trans tubulovesicular structurers andlor vesicles, since in the original characterization of this antibody, coated vesicles of the trans-tubular network were clearly immunostained (Yuan et al., 1987). No change was observed in the immunostaining pattern of the cis most Golgi cisternae in pachytene spermatocytes residing at different stages of the cycle. However, unlike in the initial report characterizing the 15C8 antibody a s a specific marker of the cis and medial Golgi cisternae, the immunostaining pattern observed in the present study is remeniscent of small vesicle immunostaining. These observations may suggest that transitional vesicles between the endoplasmic reticulum and the cis Golgi may also contain the specific protein recognized by the 15C8 monoclonal antibody. Clearly, these observations require additional ultrastructural immunocytochemical studies to verify precisely the identity of the structures immunostained with this antibody. Organelle distribution during the process of cell division must be under precise regulation to ensure that the resulting two daughter cells receive a n equal complement of each organelle. In the case of multiple copy organelles, e.g., mitochondria, a means by which cells may accomplish this distribution is to partition the or-
ganelles equally via random transport during telophase (Birky, 1983). In contrast, the Golgi apparatus is a single copy organelle (Rambourg et al., 1974, 1979, 1981) and cells must accomplish equal partitioning by a n as yet unknown mechanism, albeit one that must undoubtedly be under exact and likely complex control. For example, previous data (Robbins and Gonatas, 1964; Maul and Brinkly, 1970; Zeligs and Wollman, 1979; Hiller and Weber, 1982) and more recent evidence (Lucocq e t al., 1987) obtained in HeLa cells suggest that the Golgi apparatus undergoes a disassembly reaction prior to the onset of mitosis, resulting in the formation of small Golgi tubulovesicular clusters and free vesicles. The Golgi clusters and/or free vesicles have been proposed to serve in the partitioning of the Golgi membranes a t telophase, since they amplify the single copy Golgi complex into a multiple copy cytoplasmic organelle (Lucocq and Warren, 1987). Subsequently, the Golgi clusters appear to reassemble by a process termed “accretion of the Golgi clusters,” likely involving several intermediate steps not yet characterized fully (Lucocq et al., 1989). In addition, the endoplasmic reticulum has been implicated in the contribution of membranes to the Golgi apparatus a t telophase (Featherstone et al., 1985). Therefore, a means by which germ cells may process cytoplasmic organelles, e.g., the Golgi complex, during spermatogenesis is to recruit a mechanism similar to that evolved by somatic cells. However, our results suggest that the mechanism employed by germ cells to achieve Golgi apparatus partitioning may differ somewhat from that observed in somatic cells. First, the pachytene spermatocytes represent the longest phase of prophase of the first division of meiosis and may be observed during the first 12 stages of the cycle of the seminiferous epithelium (Leblond and Clermont, 1952). At stage 13, the pachytene spermatocytes become known a s diplotene spermatocytes and quickly terminate anaphase I and telophase I giving rise to two secondary spermatocytes. The latter exist for only a few hours and then undergo the second division of meiosis, giving rise to four round spermatids (Perey et al., 1961). Although the two meiotic divisions in themselves do not represent significant differences in the overall theme of somatic cell partitioning of organelles, it must be emphasized that the two divisions occur during a brief period of time, a t most 8 h (Perey et al., 1961). Thus, i t seems unlikely that during their short life span secondary spermatocytes are capable of synthesizing required proteins for the completion of the second division of meiosis and generate sufficient organelle-specific proteins to be subsequently partitioned into two round spermatids. Moreover, a model of somatic cell Golgi complex disassembly would suggest that intermediate size Golgi clusters should be readily detected during the second division of meiosis a s a function of time. Immunostaining of the Golgi apparatus a t this time, presumably undergoing a disassembly reaction, should give rise to a gradient in the size of the Golgi complex a s revealed by immunocytochemistry. However, the immunostaining of the Golgi apparatus of late stage spermatocytes or secondary spermatocytes revealed by the antiGIMPt antibody was not consistent with the predicted diminution in size of Golgi clusters and/or tubulovesic-
GOLGI COMPLEX OF PACHYTENE SPERMATOCYES
ular structures. Nevertheless, a rigorous ultrastructural study of changes incurred by secondary spermatocyte’s Golgi apparatus during meoisis I1 must be performed to verify our qualitative immunocytochemical results. Second, unlike somatic cells, as pachytene spermatocytes approach the first division of meiosis their Golgi apparatus increases significantly in size (Figs. 1and 2). In addition, the composition of the Golgi apparatus appears altered; the Golgi apparatus of late pachytene spermatocytes appears to contain multiple stacks of Golgi cisternae (Fig. 2). This is in marked contrast to the ultrastructural appearance of the Golgi apparatus of somatic cells that is beginning to undergo disassembly. Third, strong evidence supports a n involvement of microtubules in the process of partitioning of the small Golgi cluster once these have undergone a disassembly reaction (Allan and Kreiss, 1986). Thus, we expected to find microtubules associated with the Golgi apparatus of pachytene spermatocytes at the time of the Golgi complex Partitioning. However, unlike the Golgi apparatus in somatic cells, in our study only large Golgi apparatuses, containing several stacks of Golgi cisternae, were qualitatively observed to be associated with centrioles (Fig. a), microtubule organizing centers. These results are consistent with the recruitment of microtubules by the large Golgi apparatuses a t the time that initiation of the first division of meiosis occurs. Importantly, the results provide indirect evidence that the large Golgi apparatus may begin the process of partitioning without first undergoing a disassembly reaction. Therefore our results suggest that partitioning of the spermatocyte’s Golgi apparatus during the first meiotic division is unlikely to involve a disassembly reaction terminating in small Golgi clusters. Instead, we favor a mechanism whereby the Golgi apparatus of late pachytene spermatocytes generates several stacks of Golgi cisternae and these undergo a partitioning into the subsequent secondary spermatocytes. Whether the second meiotic division involves a dissolution of the Golgi apparatus, however, remains to be determined. Although disassembly of the Golgi apparatus into small vesicle clusters may occur during the second meiotic division, additional studies, in particular a n ultrastructural examination of secondary spermatocytes, will be required to resolve this issue. Nevertheless, because of the specific needs of the round spermatids that result from the second meiotic division it is possible to speculate that partitioning of the Golgi complex a s discrete units may accommodate the onset of spermiogenesis. Round spermatids a t stage one of the cycle enter the Golgi phase of spermiogenesis which will eventually lead to the formation of the acrosome (Clermont et al., 1988).Possibly, round spermatids minimize energy expenditures by eliminating the assembly reaction of Golgi clusters into mature Golgi apparatuses. In this regard i t must be emphasized that the generation of the acrosome is not a trivial phenomena, since the acrosome of rat spermatids eventually occupies a major portion of the cell volume. Additionally, it is not yet clear whether the Golgi apparatus is required during meiosis. For example, a structured Golgi complex present during meiosis may permit the processing of
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proteins during cell division andlor leave the newly formed round spermatids primed to begin formation of the acrosome. However, we wish to emphasize that our interpretation of the mechanism by which pachytene spermatocytes partition the Golgi apparatus is preliminary at this time and awaits verification by ultrastructural verification. Moreover, the recent studies demonstrating a role of pachytene spermatocyte products in modulating Sertoli cell function may now be understood within the context of the present data (Djakiew and Dym, 1989; Castellon et al., 1989). Increase in the size of the Golgi apparatus during pachytene spermatocyte maturation, in particular the trans most cisternae, is consistent with this observation and may provide the structural basis for the apparent increase in the secretory activity of pachytene spermatocytes. ACKNOWLEDGMENTS
We wish to acknowledge the most helpful comments of the reviewers of this manuscript. This work was supported by NIH Grants HD23484 to C.A.S.-Q. and HD16260 to M.D. LITERATURE CITED Allan, V.J., and T.E. Kreis 1986 A microtubule-binding protein associated with membranes of the Golgi apparatus. J. Cell. Biol., 2229-2239. Allison, A.C., and E.F. Hartree 1970 Lysosomal enzymes in the acrosome and their possible role in fertilization. J. Reprod. Fertil., 21.501-515. Birky, C.W. 1983 The partitioning of cytoplasmic organelles a t cell division. Int. Rev. Cytol., l5:49-89. Clermont, Y. 1956 The Golgi zone of the r a t spermatid and its role in the formation of cytoplasmic vesicles. J. Biophys. Biochem. Cyt. Suppl., 2t119-122. Clermont, Y., and X.M. Tang 1985 Glycoprotein synthesis in the Golgi apparatus of spermatids during spermiogenesis of the rat. Anat. Rec., 213.33-43. Clermont, Y., M. Lalli, and A. Rambourg 1981 Ultrastructural localization of nicotinamide adenine dinucleotide phosphatase (NADPase), thiamine pyrophosphatase (TPPase), and cytidine monophosphatase (CMPase)in the Golgi apparatus of early spermatids of the rat. Anat. Rec., 201.613-622. Clermont, Y., L. Hermo, A. Rambourg, and G. Thorne-Tjomsland 1988 Structural and cytochemical characteristics of the Golgi apparatus during the formation of the acrosomic system in r a t spermatids. In: Golgi, Lysosomes and Centriole Events in Early Spermiogenesis: Targets for Male Fertility Regulation. D. Hamilton and G.M.H. Waites, eds. Cambridge University Press, London. Djakiew, D. and M. Dym 1988 Pachytene spermatocyte proteins influence Sertoli cell function. Biol. Reprod., 39t1193-1205. Featherstone, C., G. Griffth, and G. Warren 1985 Newly synthesized G protein of vesicular stomatitis virus is not transported to the Golgi complex in mitotic cells. J. Cell Biol., 101.2036-2046. Hermo, L., Y. Clermont, and A. Rambourg 1979 Endoplasmic reticulum-Golgi apparatus relationships in the rat spermatid. Anat. Rec., 193.243-256. Hermo, L., A. Rambourg, and Y. Clermont 1980 Three-dimensional architecture of the cortical region of the Golgi apparatus in rat spermatids. Am. J . Anat., 157t357-373. Hiller, G., and K. Weber 1982 Golgi detection in mitotic and interphase cell by antibodies to secreted galactosyltransferase. Exp. Cell Res., 142t85-94. Karnovsky, M.J. 1971 Use of ferrocyanide-reduced osmium tetroxide in electron microscopy. J. Cell Biol., Abstract 284. Leblond, C.P., and Y. Clermont 1952 Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann. N.Y. Acad. Sci., 5Fit548-573 Lucocq, J.M., and G Warren 1987 Fragmentation and partitioning of the Golgi apparatus in HeLa cells. EMBO J., 6t3239-3246. Lucocq, J.M., J. Pryde, E. Berger, and G. Warren 1987 A mitotic form of the Golgi apparatus in HeLa cells. J. Cell Biol., 104.865-874. Lucocq, J.M., E.G. Berger, and G. Warren 1989 Mitotic Golgi frag-
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