Ultrastructural and immunohistochemical study of the basal apparatus of solitary cilia in the human oviduct epithelium

doi:10.1046/j.0021-8782.2001.00004.x

Journal List > J Anat > v.200(1); Jan 2002

J Anat. 2002 January; 200(1): 89–96.

doi: 10.1046/j.0021-8782.2001.00004.x.

PMCID: PMC1570880

Ultrastructural and immunohistochemical study of the basal apparatus of solitary cilia in the human oviduct epithelium

Haruo Hagiwara,¹ Shinsuke Harada,¹ Sakae Maeda,¹ Takeo Aoki,¹ Nobuo Ohwada,² and Kuniaki Takata¹

¹Department of Anatomy and Cell Biology, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371–8511, Japan

²Department of Obstetrics and Gynecology, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371–8511, Japan

Correspondence Dr Haruo Hagiwara, Department of Anatomy and Cell Biology, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371–8511, Japan. Tel.: +81 27 220 7902; fax: +81 27 220 7906; e-mail: hhagiwar/at/med.gunma-u.ac.jp

Accepted 2001.

Abstract

The basal apparatus of the solitary cilium is composed of the basal body, an associated centriole and the basal body-associated structures. To see the connection between the basal body and the centriole, we studied the basal apparatus of solitary cilia in human oviductal secretory cells by electron microscopy and immunohistochemistry. A single centriole was present in the vicinity of the basal body of a solitary cilium. The basal body and the single centriole were interconnected by one or two bundles of thin filaments with a few periodic striations. We have called these bundles the striated connector. The periodicity of striations in the striated connector measured 55 ± 6 nm, about 15 nm shorter than that of striated rootlets. The striated connector was immunolabelled with R67 antibody specific to striated rootlets, indicating that they are composed of common molecule(s). Although the true function of the connector is unknown as yet, it could play an important role for stabilising the basal body in the apical cytoplasm.

Keywords: basal body, centriole, striated connector, striated rootlet

Introduction

The centrioles are a pair of short cylinders built from nine sets of triplet microtubules (Lange & Gull, 1996). The mature mother centriole is associated with sheet-like processes and stalk-like processes (distal and subdistal appendages, respectively), whereas the daughter centriole is not (Paintrand et al. 1992). In the interphase of the cell cycle, a pair of centrioles are associated with each other by an intercentriolar link composed of long bundles of thin filaments (Paintrand et al. 1992). The electron-opaque, poorly defined osmiophilic pericentriolar material containing γ-tubulin nucleates microtubules, and serves as the microtubule organising centre (see Schiebel, 2000).

Cilia extend from the basal bodies located in the apical cytoplasm of ciliated cells. Basal bodies are duplicated through centriolar and acentriolar pathways during ciliogenesis (Hagiwara et al. 2000a). Because centrioles are structurally homologous to the basal bodies (Kellogg et al. 1994), a solitary or single cilium develops from the distal end of the mother centriole that has migrated to the cytoplasm beneath the plasma membrane (Lange & Gull, 1996). In most instances, the axoneme of solitary cilia consists of nine peripheral doublets and no central microtubules.

The basal body of solitary cilia is associated with several accessory structures including transitional fibrils, the basal foot and striated rootlets (Chakrabarti et al. 1998). Previous ultrastructural studies of solitary cilia in mammalian cells have been focused on cilia themselves and the basal body-associated structures (Wilsman, 1978; Albrecht-Buehler & Bushnell, 1980; Odor & Blandau, 1985; Baron & Salisbury, 1988; Bystrevskaya et al. 1988; Martin et al. 1988; Harrisson, 1989; Briffeuil et al. 1994; Hagiwara et al. 2000b). Little attention has been paid to the connecting structure between the basal body and the associated single centriole.

In this study, we investigated the structure that connects the basal body to its paired centriole in human oviductal secretory epithelial cells. This structure is composed of a bundle of thin filaments with cross-banding and is termed the striated connector. The periodicity of striations of the striated connector is about 15 nm longer than that of striated rootlets by morphometry. In addition, by immunofluorescence and immunoelectron microscopy with the R67 antibody against striated rootlets (Hagiwara et al. 2000b), we show that the striated connector contains a molecule common to the striated rootlets.

Materials and methods

Indirect immunofluorescence microscopy

Human oviducts were obtained with consent from women who had undergone hysterectomy and bilateral salpingo-oophorectomy due to disease of the uterus (Hagiwara et al. 2000b). The fresh oviducts were immediately embedded in OCT compound and frozen in liquid nitrogen. Frozen sections of 3 µm thickness were cut using a JUNG CM 3000 cryostat, placed on glass slides and air-dried at room temperature. The sections were fixed in 100% methanol at −20 °C for 5 min and rehydrated in PBS containing 10 mm glycine. Double immunofluorescence staining was carried out as described previously (Hagiwara et al. 2000b) using a mouse monoclonal antibody against γ-tubulin (Sigma, St Louis, MO, USA), a rat monoclonal antibody R67 against the proteins of 205/215 kDa of striated rootlets (Hagiwara et al. 2000b), FITC-conjugated goat anti-mouse IgG (Chemicon, Temecula, CA, USA) and rhodamine-conjugated goat anti-rat IgG (Chemicon). The sections were examined with an Axioplan fluorescence microscope (Carl Zeiss, Tokyo, Japan) and/or an MRC-1024ES confocal laser scanning microscope (Bio-Rad, Tokyo, Japan). Control sections were processed in parallel by replacing the primary antibody with non-immune rat serum.

Transmission and scanning electron microscopy

Human oviducts were cut into small pieces and fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 m sodium phosphate buffer, pH 7.4, for 2 h at room temperature. After washing, the specimens were processed for electron microscopy as reported (Hagiwara et al. 1997), and examined with a JEM-200CX transmission electron microscope (JEOL, Tokyo, Japan). For scanning electron microscopy, fixed oviducts were critical-point dried, coated with platinum–palladium and examined with a S-4100 scanning electron microscope (Hitachi, Tokyo, Japan).

Immunoelectron microscopy

Immunoelectron microscopic observations were carried out according to Hagiwara et al. (2000b). Oviducts were fixed in 3% paraformaldehyde in 0.1 m sodium phosphate buffer, pH 7.4, for 20 min at 4 °C and frozen in liquid nitrogen. Ultrathin frozen sections were cut, incubated with R67 for 1 h, rinsed, and treated with 6-nm colloidal gold-conjugated goat anti-rat IgG (Jackson Immunoresearch, West Grove, PA, USA). Control sections were incubated in 1% normal rat serum instead of R67. Specimens were then embedded in a mixture of methyl cellulose and uranyl acetate, and examined with a transmission electron microscope. For pre-embedding labelling of the antigen, air-dried cryostat sections of oviducts were treated with 1.0% Triton X-100 in PHEM buffer (60 mm PIPES, 25 mm HEPES, 10 mm EGTA, 2 mm CaCl₂, pH 6.9) for 10 min, washed, fixed in cold methanol and treated with R67 antibody and 6-nm colloidal gold-conjugated goat anti-rat IgG as described above. The specimens were washed in PBS, fixed with 1.25% glutaraldehyde, post-fixed with 1% OsO₄, dehydrated and embedded in epoxy resin. Ultrathin sections were cut and examined with the electron microscope.

Results

The human oviduct is lined with the simple columnar epithelium that is composed of ciliated cells and secretory cells. Solitary cilia of various length projected from the apical cell surface of secretory cells (Fig. 1). The proximal end of solitary cilia was connected to the basal body. A single centriole was located in the vicinity of the basal body (Fig. 2). More than one basal foot was usually observed at the lateral side of the basal body as reported by Odor & Blandau (1985). Both the basal body and its paired centriole were associated with striated rootlets. The rootlets were composed of bundles of longitudinal filaments of 8–12 nm diameter with periodic striations (Fig. 3). The striations consisted of dark bands of 45–50 nm thickness and interposed light bands. Fine transverse lines were visible in light bands (Fig. 3). Striated rootlets extended in various directions and ended in the cytoplasm.

Fig. 1

A scanning electron micrograph of the mucosal surface of a human oviduct. Solitary cilia (arrows) are observed on the apical surface of almost all secretory cells. Scale bar = 1.0 µm.

Fig. 2

An electron micrograph of the apical cytoplasm of a secretory cell. A solitary cilium extends from the distal end of the basal body (B). A single centriole (C) is present close to the basal body. The basal body and the centriole are connected by a short (more ...)

Fig. 3

A striated rootlet extending from the basal body (B). Note transverse lines (arrowheads) seen in each light band. Scale bar = 0.1 µm.

Basal bodies were connected to their associated centrioles by bundles of thin filaments with cross striations (Figs 4–8). Based on their location, we termed them the striated connector. As many as 50 striations were visible in striated rootlets. However, the striated connector was short and only a few striations were formed along their length. The striated connector usually linked the proximal end of the basal body to the lateral side of the centriole (Figs 4 and 5) but, occasionally, it linked the lateral side of the basal body to the one end of the centriole (Fig. 6). In a few cases, the basal body and the centriole were connected by two striated connectors (Fig. 7). The proximal end of striated rootlets was sometimes connected to the striated connector (Fig. 8). The diameter of the filaments constituting the striated connector was 6–10 nm, almost the same as that of filaments in striated rootlets. Using 30 photomicrographs of solitary cilia, we measured the periodicity of striations in striated rootlets and striated connectors, and analysed them statistically. The periodicity in the striated connector was 55 ± 6 nm, about 15 nm shorter than that of the striated rootlets (Table 1).

Fig. 4

A basal body (B) and its associated centriole (C). A fan-like bundle of thin filaments with three striations (arrows), i.e. a striated connector extends from the proximal end of the basal body to the lateral side of the centriole. Scale bar = 0.1 µm. (more ...)

Fig. 8

A basal body (B) and an associated centriole (C). The proximal end of a striated rootlet (arrowhead) is connected to a striated connector (arrows) which links the proximal end of the basal body to the centriole. Scale bar = 0.1 µm.

Fig. 5

An enlarged view of a striated connector extending from the basal body (B) to the lateral side of a centriole (C). Arrows indicate striations in the striated connector. Scale bar = 0.1 µm.

Fig. 6

A basal body (B) and its associated single centriole (C). A striated connector, striations of which are indicated by arrows, connects the lateral side of the basal body to the one end of an associated centriole. Note a striated rootlet (arrowhead) extending (more ...)

Fig. 7

A basal body (B) and its associated centriole (C). They are connected to each other by two striated connectors (arrows). Scale bar = 0.1 µm.

Table 1

Statistical analysis of the periodicity of striations in the striated connector and the striated rootlet

Immunohistochemistry with R67 decorated striated rootlets extending from the basal body/centriole region toward the interior of the cell (cf. Hagiwara et al. 2000b). Detailed examination showed that R67-positive fibrils were seen interposed at the proximal end between the basal body and the centriole (Fig. 9). This observation shows that both the striated connector and the striated rootlet were positive for R67. By immunoelectron microscopy using ultrathin frozen sections, the striated connector was decorated with immunogold particles for R67 in the same manner as the striated rootlets (Fig. 10). Labelling was prominent on the dark bands in both structures. The striated connector was also clearly labelled by gold particles by the pre-embedding method using Triton X-100-pretreated oviduct tissues (Fig. 11). Few gold particles were identified in control sections using normal rat serum instead of the primary antibody (Fig. 12). The basal apparatus of solitary cilia depicted in Figs 2–12 is schematically illustrated in Fig. 13. Three different types of striated structures are distinguished. The striated connector studied is a short filamentous bundle that generally connects the proximal end of the basal body to the lateral side of the associated centriole.

Fig. 9

Confocal micrographs of the apical cytoplasm of human oviductal secretory cells. (a,d) γ-tubulin. (b,e) R67. (c,f) Merged images of a and b, and d and e, respectively. R67-positive fibrils, namely striated rootlets, are extending from γ-tubulin, (more ...)

Fig. 10

An immunoelectron micrograph of a striated connector and a striated rootlet with R67. Gold particles are mainly seen on the dark bands of the striated connector (arrows) and the striated rootlet (arrowheads). B, Basal body; C, Centriole. Scale bar = 0.1 (more ...)

Fig. 11

An immunoelectron micrograph of a striated connector and striated rootlets with R67 by the pre-embedding method. Arrows indicate dense bands of the striated connector labelled with gold particles. Striated rootlets (arrowheads) are also labelled with (more ...)

Fig. 12

A control immunoelectron micrograph by the pre-embedding method. A striated rootlet (large arrowhead) and a striated connector (arrows) are not decorated with gold particles. Only a few gold particles (small arrowheads) are seen in the background. B, (more ...)

Fig. 13

Schematic view of the basal apparatus of a solitary cilium. The striated connector (SC) is a short cross-banded structure connecting the basal body (BB) to the centriole (C). BF, Basal foot; SR, Striated rootlet.

Discussion

The centrioles are paired in a cell. They cohere to each other by the intercentriolar link composed of long bundles of thin filaments (Bornens et al. 1987; Paintrand et al. 1992). When flagella develop from both of centrioles in algae, two basal bodies are interconnected by distal and proximal connecting fibres (Marshall & Rosenbaum, 2000). The connecting fibres disappear when the basal bodies begin to separate from each other during the early prophase of mitosis (Silflow et al. 1999). Little attention has been paid to the connecting structure between basal bodies and their associated centrioles in the cells extending solitary cilia (Ghadially, 1997). We have described in this work the striated connector that links the basal body to the single centriole. The intercentriolar link and the connecting fibre link both ends of two centrioles and both sides of two basal bodies, respectively (Paintrand et al. 1992; Marshall & Rosenbaum, 2000). The striated connector linked the basal body to the single centriole in a side to end manner. The interrelated positioning of two centriolar structures and/or the biological significance of each connecting structure might be related to the difference in the linking pattern of three connecting structures.

Solitary cilia have been considered to be immotile, because the central microtubules are lacking in the axoneme and dynein arms are hardly seen in the peripheral pairs of microtubules (for reviews see Ghadially, 1997). However, investigations using phase or videomicroscopy have revealed a vortical or funnel-like movement of solitary cilia (Odor & Blandau, 1985; Nonaka et al. 1998). The vortical movement stresses the ciliary basal apparatus in a different manner from the whip-like beating of cilia of multiciliated cells. The increase in the number of basal feet as well as structural complications of striated rootlets in the basal apparatus of solitary cilia has been considered to be related to the stabilisation of the basal body during the vortical ciliary beating (Odor & Blandau, 1985; Hagiwara et al. 2000b). By tightly connecting the basal body to its associated centriole, the striated connector may play an important role in fixing the basal body in its upright position during ciliary beating.

The distal connecting fibre in flagellated green algae contains a Ca²⁺-modulated contractile protein centrin and reorientates basal bodies during the photophobic response (McFadden et al. 1987). Thin filaments constituting the intercentriolar link are also contractile with Ca²⁺ (Paintrand et al. 1992). The contractile character of the striated connector is unknown at present. Although the striated connector and the striated rootlet are both positive for R67, the periodicity of striations in the striated connector was 15 nm shorter than that of striated rootlets. The observed shorter periodicity of striations in the striated connector might be due to the contraction of the striated connector. We will be investigating the connector by immunohistochemistry using antibodies against Ca²⁺-modulated proteins such as centrin. Additionally, in vitro experiments using the isolated solitary cilium – basal body – centriole complex will answer the question of whether the striated connector is sensitive to divalent cations and contractile.

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government, and by a grant provided by the Ichiro Kanehara Foundation.

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