THE ANATOMICAL RECORD 255:227–239 (1999) Ultrastructural and Ultracytochemical Features of Secretory Granules in the Ampullary Epithelium of the Hamster Oviduct MAJID EL-MESTRAH AND FREDERICK W.K. KAN* Department of Anatomy and Cell Biology, School of Medicine, Faculty of Health Sciences, Queen’s University, Kingston, Ontario, Canada K7L 3N6 ABSTRACT The epithelium of mammalian oviducts consists mainly of ciliated and non-ciliated secretory cells. In some mammals, secretory products originating from oviductal secretory cells have been shown to bind to the surface of, or accumulate within, ovulated eggs and/or developing embryos. These findings suggest that the secretions of the oviductal epithelial cells may play an important role in reproductive and developmental events that occur in the oviduct. In the present study, ultrastructural and cytochemical features of secretory cells in the hamster ampullary epithelium were shown by routine electron microscopy, lectin-gold cytochemistry and both conventional freezefracture and rapid-freezing techniques with special reference to the organizational aspects of their secretory granules. The use of ferrocyanide-reduced osmium tetroxide as a post-fixative in the Epon embedment of ampullary tissue samples also proved to be advantageous especially in revealing the carbohydrate contents of certain cellular compartments. The most conspicuous characteristic of the secretory cells, based on their staining property, was the presence of two types of secretory granules: those with a homogeneous electron-dense matrix and those with an electron-lucent matrix. Under favorable conditions, distinct features of the organizational arrangement of a crystalline lattice inside the secretory granules were also revealed. This well organized crystalline lattice shown in sections of Epon-embedded oviductal tissue was confirmed by examination of replicas of freeze-fractured oviducts prepared by the rapid-freezing technique. We also demonstrated with high resolution lectin-gold cytochemistry the intracellular distribution of lectinbinding glycoconjugates in the secretory cells of the hamster oviductal ampulla often in a linear array following the crystalline lattice. The results obtained in this study, taken together, provide insight into a possible link of the internal topographical features of oviductal secretory granules along with the cytochemical properties of their contents to the anticipated regulatory mechanism underlying their process of secretions. Anat Rec 255:227–239, 1999. r 1999 Wiley-Liss, Inc. Key words: oviductal glycoconjugates; oviductal ampullary epithelium; lectin-gold cytochemistry; oviductal secretion The mammalian oviduct is more than a simple tubular conduit for the transport of gametes and developing embryos. The oviductal ampulla is particularly important due to its unique role as the site of egg fertilization. In its functional state, the oviduct is also an active secretory r 1999 WILEY-LISS, INC. Grant sponsor: Medical research Council of Canada. *Correspondence to: Dr. Frederick W.K. Kan, Department of Anatomy and Cell Biology, School of Medicine, Faculty of Health Sciences, Queen’s University, Kingston, Ontario, Canada K7L 3N6. E-mail: KANFWK@POST.QUEENSU.CA Received 3 November 1998; Accepted 25 February 1999 228 EL-MESTRAH AND KAN organ that maintains and modulates a dynamic fluid-filled milieu in which many crucial developmental events occur. Oviductal fluid, which is composed predominantly of plasma derivatives (Leese, 1988) and contains secretory products that originate from the oviductal epithelium (Stone et al., 1980), provides the necessary environment for the maturation of gametes, fertilization, and early embryo development. The epithelium of the oviduct is simple columnar and consists of two main types of cells, ciliated and secretory. The non-ciliated secretory cells synthesize and secrete non-serum macromolecules or glycoproteins that are dissolved in the oviductal fluid (Oliphant, 1986). In the mammalian oviduct, glycoconjugates play an important role in mediating the interaction between male and female gametes. In some mammals, secretory products originating from the oviductal epithelium have been shown to bind to the surface of, or accumulate within, ovulated eggs and/or developing embryos (Kapur and Johnson, 1986, 1988; Léveillé et al., 1987; Kan et al., 1989; Abe and Oikawa, 1990; Boice et al., 1990; Gandolfi et al., 1991; Abe et al., 1992). Glycoconjugates can also act as hormone receptors and are essential for the successful implantation of blastocysts in the uterus (Friedricsson, 1969; Mastroianni, 1969; Menghi et al., 1985, 1986). In an earlier study, Kan et al. (1988) combined immunocytochemistry and the use of a specific monoclonal antibody against a 200-kDa oviductal glycoprotein to localize the antigenic sites exclusively to the zona pellucida of postovulatory oocytes isolated from the ampullary region of golden hamsters. Biochemical studies with monoclonal antibodies performed in the hamster demonstrated that glycoproteins of oviductal origin are detected in the ZP of postovulatory oocytes (Robitaille et al., 1988). Moreover, Araki and coworkers (1987) showed, in the hamster, a monoclonal antibody (AZPO-8) reacting with the zona pellucida of the oviductal egg but not with that of the ovarian egg. Furthermore, other reports have also indicated that embryonic development in vitro is enhanced by co-culture with oviductal epithelial cells or conditioned medium (for a review, see Bavister, 1988). These findings, taken together, suggest particular roles for the oviductal secretory cells and their secretions in reproductive and developmental events that occur in the oviduct. The ultrastructural morphology of the non-ciliated cells has been the subject of several investigations (for a review, see Ellington, 1991). Abe and Oikawa (1991) described the ultrastructural features of secretory cells in the golden hamster oviductal epithelium. However, their observations were limited to the examination of regional differences in the morphological features of oviductal secretory cells. Ferrocyanide-reduced osmium tetroxide is known to be a fixative which can enhance the staining of carbohydrate contents of certain cellular compartments (Karnovsky, 1971). Particularly, the use of ferrocyanidereduced osmium tetroxide has been shown by other investigators to allow a selective staining of the sarcotubular system, glycogen, glycocalyx as well as of unsaturated fatty acids of membranes (White et al., 1979; Aguas, 1982). The objective of the present investigation was to examine some of the ultrastructural and cytochemical features of secretory cells with special reference to the secretory granules in the ampulla of golden hamster. In this study, Epon-embedded tissue sections and freeze-fractured samples of rapidly frozen oviducts revealed the internal topography of secretory granules in the non-ciliated secretory cells. These data correlated with results obtained with lectin cytochemistry to show the distribution of different carbohydrate residues in relation to the internal organization of the secretory granules. The use of ferrocyanidereduced osmium tetroxide allowed a better distinction between two different types of secretory granules based on the staining property of their carbohydrate contents. On the basis of these observations, we suggest that the unique internal topography of both types of secretory granules, added to their differential staining and cytochemical properties, may reflect a difference in the secretory products of ampullary secretory cells as well as a possible link of such topography to the anticipated mechanism regulating their secretions. MATERIALS AND METHODS Preparation of Oviductal Tissue Eighteen sexually mature (8 to 12 weeks) female golden hamsters (Mesocricetus auratus) (Charles River, StConstant, Quebec, Canada) were housed over hardwood bedding (Beta chip; Northeastern Products Corp., Warrensburg, NY). The hamsters were maintained on a 12-hr light/dark cycle, and were freely provided with food (Purina Rodent Chow; Ralston Purina International, Strathray, Ontario, Canada) and drinking water. They were acclimatized to laboratory conditions for at least 7 days before experimental procedures were initiated. To obtain oviductal tissue, six of the animals designated for cytochemical study were killed by cervical dislocation. Their ventral abdominal wall was immediately cut open and the oviducts were excised, washed briefly with phosphatebuffered saline (PBS), pH 7.4, and examined under a dissecting microscope to localize the ampullary region of the oviducts. The ampullary portions were then fixed for 2 hr at 4°C by immersion in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). For post-embedding labeling, the ampullary tissue samples were trimmed into small cubes and then dehydrated in a series of graded methanol solutions, infiltrated, and embedded in Lowicryl K4M according to routine procedure. The ampullary tissue samples of another six animals, designated for morphological study only, were post-fixed with 1% osmium tetroxide in nanopure water for 2 hr at 4°C following glutaraldehyde fixation by immersion as detailed above. Some of the tissue samples were treated with the ferrocyanide-reduced osmium tetroxide method (Karnovsky, 1971). Post-fixation with the latter method was carried out at 4°C for 2 hr. In either case, between the fixation and post-fixation, tissue samples were washed three times in 0.1 M cacodylate buffer, trimmed into small cubes, and after post-fixation they were dehydrated in a series of graded ethanol solutions, infiltrated, and embedded in Epon 812. Sections of Epon- and Lowicryl-embedded tissue samples measuring 1 µm in width were first examined by light microscopy in order to locate the area of interest. Lowicryl K4M thin sections of pale gold interference color were then cut with glass knives on a LKB ultramicrotome and mounted on 200 mesh nickel grids having a formvar-carbon-coated film. Epon sections were cut with a diamond knife and were mounted on copper grids having a formvar-carbon-coated film. SECRETORY GRANULES IN OVIDUCTAL AMPULLA Cytochemical Labeling For lectin cytochemistry, colloidal gold was used as a marker. Cytochemical labeling was performed by the one-step and two-step postembedding methods as described by Benhamou (1986). The one-step method (direct labeling) was performed with HPL and RCA I lectins, and the two-step method (indirect labeling) with WGA and LFA lectins. Preparation of Colloidal Gold, Lectin-Gold and Glycoprotein-Gold Complexes Colloidal gold particles of 15 nm diameter were prepared by the sodium citrate method as described by Frens (1973). The lectins used were Helix pomatia lectin (HPL), specific for N-acetyl- D-galactosamine (Gal NAc) (Hammarstrom and Kabat, 1969, 1971); wheat germ agglutinin (WGA), specific for N-acetylglucosamine (GlcNAc) (Nagata and Burger, 1974)/sialic acid (Peters et al., 1979); Limax flavus agglutinin (LFA), specific for sialic acid (Miller et al., 1982); and Ricinus communis agglutinin I (RCA I), specific for D-galactose (Olsnes et al., 1974; Irimura et al., 1975). Direct Helix pomatia lectin-colloidal gold (HPL-CG) complex and direct Ricinus communis agglutinin I-colloidal gold (RCA I-CG) complex were prepared as described by Roth (1983). Ovomucoid-gold complex and fetuin-gold complex were prepared as described by Geoghegan and Ackerman (1977) and Roth et al. (1984), respectively. Ovomucoid, fetuin, HPL, WGA, and RCA I were purchased from Sigma Chemical Co. (St. Louis, MO) and LFA from Terochem Lab Ltd. (Mississauga, ON). Cytochemical Controls The labeling specificity of both HPL-CG and RCA I-CG was assessed by incubating the tissue sections at room temperature in presence of their blocking sugars, GalNAc (0.2 M) and D-galactose (0.2 M), respectively. For LFA, the labeling specificity was assessed as follows: 1) sections were incubated at room temperature with LFA in presence of its blocking sugar sialic acid (0.2 M) followed by incubation with fetuin-gold complex; 2) sections were digested with neuraminidase for 2 hr at 37°C prior to labeling with LFA followed by incubation with fetuin-gold complex; 3) sections were incubated at room temperature with fetuingold complex alone. For WGA, the following controls were used: 1) sections were incubated at room temperature with WGA in presence of its blocking sugars GlcNAc (0.1 M) and sialic acid (0.1 M) followed by incubation with ovomucoidgold complex; 2) sections were incubated at room temperature with ovomucoid-gold complex alone; 3) sections were incubated at room temperature with WGA in presence of its blocking sugar Glc NAc (0.2 M) followed by incubation with ovomucoid-gold complex. Freeze-Fracture Procedure Four mature female hamsters were used for routine freeze-fracture studies. For freeze-fracture, the animals were sacrificed by cervical dislocation. The oviducts were excised as described above and fixed immediately by immersion in 2.5% glutaraldehyde in 0.1 M cacodylate buffer. While in fixative, the ampullary region was identified and isolated under a dissecting microscope. The ampullary tissue samples were further fixed for 2 hr at 229 4°C, washed with 0.1 M cacodylate buffer and then infiltrated with 25% glycerol in 0.15 cacodylate for at least 1 hr at 4°C. Blocks of tissue samples were mounted on Balzers type gold disks and frozen in liquid nitrogen cooled Freon 22. Freeze-fracture was carried out in a Balzers unit at ⫺130°C under a vacuum of 2 ⫻ 10⫺6 Torr without etching. This was followed by shadowing with platinum (Pt) at a fixed angle of 45° and coating with carbon (C) at a 90° angle. The thickness of the replica was approximately 2 nm Pt and 25 nm C as determined by a Balzers crystal thin-film monitor. In addition to routine freeze-fracture study, two other animals were sacrificed in the same manner and the ampullary portions of the oviducts were isolated and mounted immediately on Balzers gold disks without fixation and cryoprotection. The freshly isolated, unfixed tissue samples were rapidly frozen at ⫺170°C in a rapidfreezing device (Life Cell). The rapid-frozen samples were then freeze-fractured in a Balzers unit at ⫺130°C and etched for 3 min at ⫺100°C before replication was made as described above. Platinum-carbon replicated specimens prepared by both routine freeze-fracture method and rapid-freezing technique were digested in sodium hypochlorite to remove the tissue debris. The replicas were then washed three times in bidistilled water and mounted on 300 mesh copper grids before being examined on an electron microscope. RESULTS Ultrastructural Morphology of the Ampullary Epithelium of the Hamster Oviduct Electron microscope examination of the ampullary epithelium showed the presence of two main types of cells, ciliated cells and non-ciliated secretory cells. The ciliated cells possessed long cilia interspersed between microvilli at their apical cell surface (Fig. 1). Mitochondria and membrane-bound lysosome-like vesicles were frequently seen in the supranuclear region. Secretory cells could be identified by the absence of cilia at their cell surface (Fig. 1). The most conspicuous characteristic of the secretory cells, however, was the presence of secretory granules of variable sizes and distinct staining properties (Figs. 1 and 2). The intracellular location of granules was typically restricted to the supranuclear and apical cytoplasm although a few isolated granules could be observed elsewhere in the cytoplasm, including the basolateral compartment. Immature secretory granules, with their contents showing a granular-like appearance in cross section, were commonly located in the vicinity of the Golgi zone (Fig. 2). Many coated vesicles were also seen associated with the Golgi saccules (Fig. 2). Occasionally, well-preserved and favorably cut sections of the ovoid and, presumably, more mature secretory granules located at the apical region of secretory cells revealed their highly organized contents. The latter appeared in the form of parallel, lamella-like structures with one of their two ends radiating from a round dense core located at one pole of the granule and with their other ends converging to meet at the opposite pole (Fig. 3a). In secretory granules with a different plane of cut, cross-sectioned profiles and oblique view of the lamella-like structures constituting the contents of the granules were also revealed (Fig. 3b). The internal organization of the secretory granules were further examined using platinum/carbon coated replicas 230 EL-MESTRAH AND KAN Figs. 1, 2. Electron photomicrographs of thin-sections of Eponembedded hamster oviductal ampulla. Ciliated cells (CC) are characterized by the presence of cilia (Ci) shown in cross-section at the apical cell surface with mitochondria (M) distributed in clusters in the apical region of the cell. Secretory cells (SC) are characterized by the presence of numerous secretory granules (SG) at their apical pole (Fig. 1) and their cytoplasm appears to be more compact and darkly stained. Secretory cells also possess a well-developed Golgi apparatus (Gol) made up of six to eight saccules frequently seen to be associated with secretory granules (SG) (Fig. 2). Note the presence of the many coated vesicles that are associated with the Golgi saccules (arrowheads) (Fig. 2). Note also the slight difference in the staining property of secretory granules (SG1 and SG2) in Figure 2. Nu, nucleus; Lu, lumen. Fig. 1, ⫻16,000; Fig. 2, ⫻25,500. SECRETORY GRANULES IN OVIDUCTAL AMPULLA 231 Fig. 3. a: A favorably cut section of a secretory granule with its internal distinct lamella-like structures in beaded-chain appearance (arrowheads) are seen radiating from a small and round dense core (arrow) at one pole of the secretory granule. b: A secretory granule sectioned in a different plane showing cross-sectioned profiles (asterisk) of the lamellae. a, ⫻16,900; b, ⫻18,200. prepared by both routine freeze-fracture and rapidfreezing techniques. In replicas prepared from frozen samples of glutaraldehyde-fixed and cryoprotected oviductal tissue, many cross-fractured profiles of secretory granules were seen (Fig. 4). The contents of crossfractured granules had a relatively smooth appearance with a very fine granular texture (Fig. 4a). Parallel arrays of lamella-like profiles corresponding to those seen in Epon sections were not seen in routine freeze-fracture preparations. However, when unfixed and rapidly frozen samples were fractured and etched followed by shadowing with platinum and carbon, the resulting replicas revealed both longitudinally and cross-fractured profiles of lamella-like lattice in the secretory granules corresponding to similar structures observed in Epon sections (Figs. 4b and c). The post-fixation of tissue samples with ferrocyanidereduced osmium tetroxide provided the opportunity for a better demonstration of the morphology of the secretory cells. The darkly stained cytoplasm of the secretory cells was typical when compared to that of the ciliated cells (Fig. 5). The secretory granules were usually of two types: 1) those with a homogeneous electron-dense matrix and 2) those with an electron-lucent matrix. The heavily stained secretory granules were easily distinguishable from those displaying a lesser degree of opacity. Both types of granules appeared to occur within the same cell type (i.e. in secretory cells) and possessed a relatively small dense core (Fig. 6a). In both cases, the dense core was seen to be preferentially located at one pole of the secretory granule and appeared to be in contact with the granule membrane. Some of these granules appeared to have coalesced into a large expanse (Fig. 6b). The release of granule content to the ampullary lumen at the apical surface of the nonciliated cells and cytoplasmic protrusions were frequently observed. The glycocalyx covering the microvilli was heavily stained due to its glycoprotein content, whereas the cilia possessed no such appearance. Electron microscopic observation of the ampullary secretory cells revealed, in the cytoplasm, the presence of a well developed Golgi complex with elaborate organization (Fig. 7). Electron-lucent secretory granules were always found in the trans-face of the Golgi complex whereas densely stained granules were found in both the trans- and cis-face of the Golgi apparatus. Some palely stained secretory granules displayed cross-sectioned profiles of the lamella-like structures previously seen in Epon-embedded oviduct tissue without prior treatment with ferrocyanide-reduced osmium tetroxide (Fig. 7a). In addition, many small vesicles of uniform size were also found in the vicinity of the Golgi apparatus (Fig. 7a). The small round vesicles were located between cisternae of endoplasmic reticulum and the cis-face of the Golgi apparatus, between the ends of stacks of Golgi saccules and also concentrated in an area close to the trans-face of the Golgi complex. Most of the cisternae of endoplasmic reticulum adjacent to the cis-face of the Golgi complex appeared to be of the smooth type. However, individual endoplasmic reticulum cisternae studded with ribosomes on one surface were also seen in the neighbourhood of the cis-face of the Golgi complex. Occasionally, a row of these small, uniform-sized vesicles were found to lie adjacent to the first cis Golgi saccule (Fig. 7a). In some Golgi stacks, a gradient of staining intensity was seen in the saccules with a progressive increase in staining intensity from the cis-face towards the trans-face (Fig. 7b). Fig. 4. Electron photomicrographs of freeze-fractured oviductal ampulla prepared by both conventional freeze-fracture (a) and rapid-freezing techniques (b,c). a: Cross-fractured secretory granules (SG) in secretory cells of oviductal ampulla prepared by the routine freeze-fracture method show their contents with a relatively smooth texture (asterisk). However, when rapidly frozen ampullary samples were fractured and etched, the resulting replicas revealed both longitudinally (arrowheads) and cross- fractured (asterisk) profiles of the lamella-like lattice occupying the matrix of the secretory granules (SG) (b,c); Such lamella-like profiles correspond to similar structures seen in Epon sections. Note that the presence of both longitudinally and cross-fractured profiles can be seen within the same secretory granule (SG) as a result of the fracturing process (c). a, ⫻26,000; b, ⫻29,900; c, ⫻27,200. Fig. 5. Electron photomicrograph of Epon-embedded ampulla previously post-fixed with ferrocyanide-reduced osmium tetroxide. The columnar-shaped ciliated cells (CC) have a palely stained cytoplasm with the nucleus (Nu) located basally. However, secretory cells (SC) characterized by the presence of microvilli (Mi) at their apical surface and the assembly of numerous secretory granules (SG) at their apical pole are seen protruding into the ampullary lumen (Lu). Note the presence of two types of granules in the secretory cells (SC); the electron-dense secretory granules (SG) are much more abundant than those which are electronlucent (asterisk). Ci, cilia; M, mitochondria. ⫻12,500. Fig. 6. Electron micrographs showing the secretory granules of the ampullary secretory cells at high magnifications (a). The two types of secretory granules (SG) are easily identifiable due to their differential staining; a dense core (arrowheads) is located at one pole of each of both types of secretory granules. b: A secretory granule (SG) is seen releasing its content through the free edge of the apical cell surface (arrowheads) into the ampullary lumen (Lu), whereas other secretory granules appear to coalesce to form a larger expanse (asterisk) . a, ⫻25,000; b, ⫻30,000. SECRETORY GRANULES IN OVIDUCTAL AMPULLA Fig. 7. Electron photomicrographs showing the Golgi region of secretory cells in the oviductal ampulla previously treated with ferrocyanidereduced osmium tetroxide prior to Epon embedment. a: A well-developed Golgi apparatus (Gol) is seen embracing both electron-dense (asterisk) and electron-lucent (SG) secretory granules. Their contents reveal cross-sectioned profiles (thick arrows) of beaded chains of lamella-like structures previously seen cut longitudinally in Figure 3a. Note the 235 presence of several darkly stained granules located near both the cis-face and trans-face of the Golgi complex, and the presence of a row of small uniform-sized vesicles adjacent to the cis-face of the Golgi stack (arrowheads). b: A Golgi apparatus (Gol) showing a gradient of staining intensity increasing progressively from the cis- to the trans-face; note also the presence of a fenestrated saccule (arrowheads) at the trans-face of the Golgi apparatus (Gol). a, ⫻42,500; b, ⫻43,000. 236 EL-MESTRAH AND KAN Cytochemical Localization of Lectin-Binding Glycoconjugates in the Hamster Oviductal Ampulla In the present study, we also examined the distribution of lectin-binding glycoconjugates in relation to the contents of the granule in Lowicryl-embedded tissue sections of the ampullary epithelium. For this purpose, four different lectins were used. Quantitative results of cytochemical labeling with various lectins have been detailed in a previous study (El-Mestrah and Kan, 1999). Since the cytochemical features of secretory granules are of major interest in this study, special attention was directed toward the distribution of gold particles in the secretory granules of oviductal ampulla with the possible correlation of such distribution to their internal morphology. Although the use of Lowicryl K4M hydrophilic resin for lectin cytochemistry compromised the ultrastructure of the cells, tissue sections incubated with each of HPL-CG (Figs. 8a and b), RCA I-CG (not shown), LFA (Fig. 8c), and WGA (not shown) presented similar patterns of labeling by gold particles. The distribution of gold particles observed over the secretory granules of non-ciliated secretory cells is best illustrated by electron micrographs taken from ampullary sections incubated with HPL-CG (Figs. 8a and b) and LFA (Fig. 8c). The labeling of lectin-binding glycoconjugates in some secretory granules appeared to be aligned with the lamella-like lattice occupying the matrix of the granules (Figs. 8b and c) with no sign of labeling over the dense core (Fig. 8a). Occasionally, labeling by gold particles could be seen associated with the secretory products in the process of their release into the oviductal lumen (Fig. 8b). On the other hand, ciliated cells which are characterized by the presence of long cilia interspersed between microvilli at their apical cell surface, showed no indication of any labeling in their cytoplasm and its associated organelles except for a weak labeling over the ciliary extensions in the ampullary lumen (not shown). Control incubations of HPL, RCA I, and LFA (not shown) showed negative reaction to the corresponding lectin-gold complex demonstrating the specificity of the labelings. Tissue sections incubated with WGA in presence of its blocking sugars GlcNAc and sialic acid or with ovomucoidgold complex alone were also negative (not shown). DISCUSSION The mammalian oviduct is a secretory organ which provides the environment necessary for the maturation and transport of gametes and embryo development. In particular, the oviductal ampulla has a fundamental importance owing to the fact that it is the site where fertilization takes place. The ultrastructural morphology of the oviduct is closely related to its functions. In the present study, we examined the ultrastructure and cytochemical properties of secretory cells of the ampullary epithelium in the golden hamster using various techniques in electron microscopy. One distinct feature of the oviductal ampulla observed in this study was the ultrastructural appearance of secretory granules. The morphological features of secretory granules of oviductal secretory cells have been investigated in several mammalian species including the human. It has been suggested that two types of secretory granules, namely, electron-dense and electron-lucent granules, are present in the oviductal secretory cells of the mouse (Komatsu and Fujita, 1978), golden hamster (Abe and Oikawa, 1991), rabbit (Brower and Anderson, 1969; Jansen and Bajpai, 1982), cow (Abe et al., 1993), monkey (Odor et al., 1983), and human (Björkman and Fredricsson, 1962; Clyman, 1966), while Willemse and Van Vorstenbosch (1975) demonstrated the presence of four types of secretory granules in the sheep oviduct. In a previous study performed in the hamster oviduct, Abe et al. (1991) demonstrated the presence of regional differences in the ultrastructural features of secretory cells and their related secretory granules. These various studies, taken together, have shown that there are marked differences among species as well as regional differences in the morphological features of secretory granules. In the present study, favorable Epon sections of the secretory granules revealed their distinctive internal organization whereby well organized lamella-like structures appeared to be radiating from a dense core located at one end of the granule; this finding may suggest a possible role for the dense core in the secretory mechanism of the secretory granule. However, the unknown nature and the functional role of the dense core as well as that of the lamella-like structures need to be further investigated. The dense core found located at one pole of some secretory granules was not present in all secretory granules. This is possibly due to the plane of cut. However, in the golden hamster, dense cores were rarely seen in the immature secretory granules, but they appeared during the maturation process of secretory granules (Abe and Oikawa, 1989). Similar phenomena were observed in the sheep (Willemse and Van Vorstenbosch, 1975). These findings suggest that the presence of dense cores is a characteristic of mature secretory granules in the oviduct. Furthermore, some secretory granules which were located in the vicinity of the Golgi apparatus, and are therefore considered immature, showed cross-sectioned profiles of lamellar-like structures similar to those of the mature ones, suggesting that these immature granules might have attained their actual internal topography early on during the process of maturation. Although two types of secretory granules appeared to occur within the non-ciliated secretory cells, a distinction between the two types was made more evident with the use of ferrocyanide-reduced osmium tetroxide by which the secretory granules were shown to be differentially stained. This is suggestive of the presence of a heterogeneous population of secretory granules in the hamster oviductal ampulla and that the secretory granules might contain different secretory contents. Although the presence of similar lamella-like lattice in the oviduct secretory granules has been reported in sevFig. 8. Electron photomicrographs of the ampullary epithelium labeled with HPL-gold (a,b) and with LFA and fetuin-gold complexes (c). a: A high concentration of gold particles is localized over the secretory granules (SG), Golgi apparatus (Gol) and microvilli (Mi) of a secretory cell. Note that the dense core (thick arrows) occupying one pole of the secretory granules (SG) is devoid of any labeling. b: Colloidal gold particles are uniformly distributed throughout the secretory granules (SG) which appear to have coalesced. The labeling by gold particles is shown here to be aligned with the lamella-like profiles (arrowheads) occupying the matrix of the secretory granules (SG) during the secretory process. c: A region of a secretory cell from a tissue section incubated with LFA and fetuin-gold complex showing secretory granules (SG) heavily labeled with gold particles. In this micrograph, gold particles can be seen superimposed over the lamella-like structures (arrowheads) of the secretory granules (SG) . Lu, lumen; Nu, nucleus. a, ⫻34,000; b, ⫻47,000; c, ⫻17,000. SECRETORY GRANULES IN OVIDUCTAL AMPULLA Figure 8. 237 238 EL-MESTRAH AND KAN eral other animal species (Nayak and Ellington, 1977; Odor et al., 1983), results obtained in our study have provided, perhaps, the best view of the arrangement of such lamella-like structures in the granules at the ultrastructural level. Using the rapid-freezing and freeze-etch techniques on freshly isolated, unfixed oviductal tissue, we have been able to confirm the distinct entity of these lamella-like structures shown in thin sections of Eponembedded oviducts. In the present study, as shown by results obtained with both the routine osmium tetroxide post-fixation Eponembedded specimens and tissue treated with ferrocyanidereduced osmium tetroxide, the Golgi apparatus in the non-ciliated cells of the hamster oviduct is a highly elaborate organelle typical of secretory cells. The small round vesicles that were observed in between the endoplasmic reticulum cisternae and the first cis Golgi saccules are likely transport vesicles shuttling newly synthesized proteins from the endoplasmic reticulum cisternae to the forming (cis) face of the Golgi complex. The progressive increase in staining intensity of the Golgi saccules from the cis-face to the trans-face (Fig. 7b), as shown by ferrocyanide-reduced osmium tetroxide impregnation, reflects an increase in the carbohydrate moieties associated with newly synthesized macromolecules in the secretory cells during their processing through the Golgi stacks. It is interesting to note that small coated vesicles were being budded off the edges of the Golgi saccules and that some of these coated vesicles could be seen to deliver their content to immature secretory granules in formation (Fig. 2). It appears that some of the coated vesicles that pinched off from the ends of the Golgi saccules were being diverted to a specific destination (formation of secretory granules) other than the plasma membrane in the hamster oviductal secretory cells. The high resolution cytochemistry has allowed for the localization of lectin-binding glycoconjugates in intracellular organelles involved in their biosynthesis and secretion. The high density of labeling for HPL-, WGA-, LFA-, and to a lesser extent RCA I-binding glycoconjugates over the Golgi apparatus and secretory granules indicates an active synthesis and secretion of the products by the ampullary non-ciliated secretory cells. In a previous study performed in the hamster, we demonstrated by lectin-gold cytochemistry that HPLbinding glycoconjugates are absent from the zona pellucida (ZP) of ovarian oocytes but are synthesized and secreted by the non-ciliated secretory cells in the oviduct and later transferred to the zona pellucida of ovulated eggs (Kan et al., 1990). Subsequently, we demonstrated, with the high resolution lectin-gold approach and quantitative analysis, changes of glycoconjugates in the hamster ZP during oocyte growth and development in the ovary and oviduct (Roux and Kan, 1991). Similarly, previous studies from other laboratories demonstrated the distribution of lectin-binding glycoconjugates in relation to the estrous cycle (Vrcic, 1993; Raychoudhury et al., 1993; Wu et al., 1993; Menghi et al., 1985). Together, these results suggest that the oviductal epithelium is a bona fide site for the synthesis and secretion of glycoconjugates that may be involved in many developmental events occurring in the oviduct. One of the aims of our study was to draw a correlation between the distribution of lectin-binding glycoconjugates in the secretory granules and their internal structure. In the present study, cytochemical labeling with the various lectins (in particular, HPL and LFA) showed that the gold particles were directly located over the crystalline lattice in the granule matrix. The organization of lamella-like structures within the secretory granules may serve as an anchor for glycoconjugates prior to their release into the ampullary lumen, or the glycoconjugate macromolecules themselves may be anchored to each other in a highlyorganized manner. The absence of labeling over the dense core of secretory granules requires further investigation in order to unravel its chemical composition and its possible role in the secretory process. In summary, we have revisited the ultrastructure and cytochemical properties of secretory granules in the ampullary region of the golden hamster using a combination of routine electron microscopy, freeze-fracture techniques and lectin-gold cytochemistry. We have provided new ultrastructural information on the internal organization of the ampullary secretory granules and the structural entity of the crystalline lamella-like lattice decorated by lectinbinding glycoconjugates as demonstrated in the present study. With the application of the ferrocyanide-reduced osmium tetroxide staining procedure, we have been able to elucidate the heterogeneity of secretory contents based on the differential staining properties of the secretory granules. The ampullary region of the female reproductive tract is a strategic site in the complex process of fertilization since it is in the lumen of the ampulla where the sperm fertilizes the egg. The ampulla plays a vital role in this process by contributing its secretion to the luminal environment. Therefore, the revelation of the internal topographical features of oviductal secretory granules, at both morphological and cytochemical levels, allowed us to raise some valid speculations on the possible correlation of such internal organization to the regulatory mechanism underlying their secretory process. In the future, it would be necessary to unravel the precise role of the dense core in the secretory mechanism as well as the function of the highly organized secretory contents within the membrane bound granules. ACKNOWLEDGMENTS The authors wish to thank Mr. Bob Temkin and Ms. Verna Norkum for reproduction of the original photomicrographs. LITERATURE CITED Abe H, Oikawa T. 1989. 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