15Proc. Arthropod. Embryol. Soc. Jpn. 52, 15–18 (2019)Ⓒ 2019 Arthropodan Embryological Society of Japan
ISSN 1341–1527[SHORT COMMUNICATION]

Histological Study of the Ovarian Follicle Epithelium in Hyleoglomeris(Diplopoda, Glomerida, Glomeridae): Two Subpopulations in theFollicle Epithelial Cells
Yasuhiko CHIKAMI1, 2) and Kensuke YAHATA3)
Graduate School of Life and Environmental Sciences, University of Tsukuba, 1–1–1 Tennodai, Tsukuba, Ibaraki 305–8572, Japan
Current address: Department of Basic Biology in the School of Life Science, SOKENDAI, Nishigonaka 38, Myodaiji, Okazaki, Aichi
444–8585, Japan
3)
Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, 1–1–1 Tennodai, Ibaraki 305–8572, Japan
E-mail: yahata@biol.tsukuba.ac.jp (KY)1)
2)

The oocytes of most arthropods are surrounded by ovariansomatic tissue, i.e., follicle epithelium (Chelicerata: Makioka,1979; Myriapoda: Yahata et al. 2018; Crustacea: Kubrakiewicz
et al., 2012; Hexapoda: Büning, 1994). The follicle epitheliumof the Hexapoda contains subpopulations of cells (Margaritiset al., 1980; Büning, 1994; Garbiec and Kubrakiewicz, 2012):
for example, in Drosophila melanogaster, 11 subpopulations offollicle epithelial cells, such as posterior polar follicle cells, canbe detected (Büning, 1994). These subpopulations of follicle
epithelial cells differ from each other in their morphologicalfeatures, i.e., cell-shape, organelle composition, and spatial
position within the follicle epithelium (Margaritis et al., 1980;
Tworzydlo and Kisiel, 2011), and have special functions that areimportant for oogenesis such as choriogenesis (Margaritis etal., 1980; Büning, 1994).
Although a number of studies have investigated thesubpopulations of hexapod follicle epithelial cells (Büning,1994; Tworzydlo and Kisiel, 2011), the cellular subpopulationalconstruction of the follicle epithelium is yet to be clarified inarthropods other than Hexapoda. We discovered the cellularsubpopulational construction of the follicle epithelium in
two Hyleoglomeris (Diplopoda: Glomerida) species, andthe histological and histochemical characteristics of these
subpopulations of the cells are detailed herein.
Adults of Hyleoglomeris japonica Verhoeff, 1936 werecollected in Mito and Tsukuba, Ibaraki Prefecture from May toOctober 2014 and from April to July 2015. Adults of Hyleoglomeris
yamashinai Verhoeff, 1937 were collected in Uruma, Okinawa
Prefecture in September 2014. Their ovaries were fixed eitherwith Bouinʼs solution overnight, with 4% paraformaldehyde
for 3 h or with Karnovskyʼs solution (2% paraformaldehyde+ 2.5% glutaraldehyde) for 2 h. Some fixed specimens
were dehydrated in a graded ethanol-n-butanol series andembedded in paraffin while others were dehydrated in a gradedethanol series and embedded in methacrylate resin (Technovit7100: Külzer, Wehrheim, Germany). The 5-µm-thick paraffin
sections and the 2-µm-thick resin sections were stained with
Mayerʼs hematoxylin–eosin and then observed under a lightmicroscope (BH-2: Olympus, Tokyo, Japan). To observe cells,
the fixed ovaries were stained with rhodamine phalloidin for 40
min and 1 µmol/l 4ʼ,6-diamidino-2-phenylindole (DAPI) for 30
min, and observed under a fluorescence microscope (Eclipse
Ni-U: Nikon, Tokyo, Japan) with excitation at 540 and 360 nm.
The H. japonica and H. yamashinai oocytes weresurrounded by a flattened, single-layered follicle epithelium,
which was connected to the ovarian epithelium. The follicle
opened into the hemocoel at the follicle pore (Fig. 1A, D). The
gross morphology of the ovaries in these species, as observedin the present study, was identical to that reported in previous
studies of H. japonica (Yahata and Makioka, 1997) and othermyriapods (Yahata and Makioka, 1994; Miyachi and Yahata,2012; Yahata, 2012; Yahata et al., 2018).
In the Hyleoglomeris species examined, the follicle
epithelial cells measured 1–2 µm in height while the cells
opposite the follicle pore measured 2–10 µm in height (Figs.
1B, C, E, 2A). The epithelial cells opposite the follicle pore
were therefore thicker than those located elsewhere (Fig.
1A, D) and could clearly be distinguished from the thinner
cells according to their location within the follicle. The
follicle epithelium of Hyleoglomeris was thus composed of two
subpopulations of cells which can be distinguished by the cellshape and spatial position within the follicle epithelium. The
follicle epithelial region opposite the follicle pore composed ofthicker cells is herein referred to as the “apical thick region,”while the region composed of thinner cells is referred to as the“main-body region.” Among Arthropoda, structurally different
subpopulations of follicle epithelial cells have been described
only in Hexapoda (Büning, 1994): hence, this is the first reportof such subpopulations of follicle epithelial cells in arthropods
other than Hexapoda.

16Y. CHIKAMI AND K. YAHATA

In the apical thick region, 10 or more cells (Fig. 2A, Aʼ)
were aligned in a rosette-like formation (Fig. 2B, Bʼ) with their
nuclei located opposite to the center of the rosette (Fig. 2Bʼ).
The cells of the apical thick region were more eosinophilic thanthose of the main-body region (Fig. 1A, C–E). As acidophilicmaterials are known to show stainability to eosin, it is suggested
that the apical thick region is rich in acidophilic materials suchas proteins. Additionally, the cells of this region contained
many lipid droplet-like vacuoles (Fig. 1C, E). These featuresindicate that the apical thick region has a secretory function.
Strong phalloidin staining was also observed in this region(Fig. 2Aʼ), especially in its central area (Fig. 2Bʼ), suggestinga high inclusion of F-actin. Specific F-actin localization has
previously been demonstrated in specialized subpopulations

Fig. 1 The ovarian follicles and follicle epithelia of Hyleoglomeris japonica and H. yamashinai. A. The ovarian follicle of H. japonica showing
two distinct regions, i.e., the apical thick region and the main-body region. Paraffin. B. Main-body region of H. japonica follicleepithelium. The main-body region constructs a major part of follicle epithelium with cells measured 1–2 µm in height. Methacrylate
resin. C. Apical thick region of H. japonica follicle epithelium. The apical thick region is composed of follicle epithelial cells measuring
2–10 µm in height. The cells of this region intensely stained by eosin, are rich in lipid droplet-like vacuoles (arrowheads). Methacrylate
resin. D. The ovarian follicle of H. yamashinai. As in H. japonica, the apical thick and main-body regions of the follicle epithelium
can be distinguished. Paraffin. E. Detail of the apical thick region of the H. yamashinai follicle epithelium. Paraffin. atr: apical thickregion, fp: follicle pore, hc: hemocoel, mbr: main-body region, oe: ovarian epithelium, ol: ovarian lumen, oo: oocyte, arrowhead: lipid
droplet-like vacuole. Scales = A, D: 50 µm; B, C, E: 10 µm.

OVARIAN FOLLICLE EPITHELIUM IN HYLEOGLOMERIS

17Fig. 2 Whole-mount images of the ovarian follicle of Hyleoglomeris japonica A, Aʼ. DIC (A) and fluorescence microscope (Aʼ) images of theapical thick region of the follicle epithelium, stained by phalloidin, lateral view. B, Bʼ. DIC (B) and fluorescence microscope (Bʼ) imagesof the apical thick region of follicle epithelium, stained by phalloidin and DAPI, apical view. atr: apical thick region, mbr: main-bodyregion, oo: oocyte. Scales = 50 µm.

of epithelial cells in the follicles of hexapods (Blattodea:Zhang and Kunkel, 1992; Diptera: Büning, 1994; Phthiraptera:Zawadzka et al., 1997). These specialized subpopulations ofthe follicle epithelial cells with high F-actin levels are known
to play important roles in oogenesis, such as formation of themicropyle (e.g., Zhang and Kunkel, 1992; Kubrakiewicz et al.,2005; Zawadzka et al., 1997). F-actin richness in the apical thickregion in Hyleoglomeris suggests that it has a possible special
function related to oogenesis.
To fully understand the apical thick region in Hyleoglomeris
follicles, intensive ultrastructural, histochemical anddevelopmental studies are needed, which will demonstrate
the diversity of ovarian somatic tissue in myriapods andarthropods.

Shibata, Ms. Y. Takatani and Mr. N. Naya for their assistancewith sample collection. We sincerely thank the editorial
secretary and two reviewers who gave us kind suggestions.
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