VENUS 65 (4): 345-353, 2007

Notes on the Histology of Ovarian Eggs of the Epipelagic Octopus Ocythoe tuberculata Caught off Naoetsu Port, Niigata Prefecture, Sea of Japan — Studies on Gonad Maturity in Some Marine Invertebrates－XIII —

Yoshiharu Honma1*, Tatsuo Ushiki1, Masaei Takeda1 and Yukihiro Nakamura2 1Division of Microscopic Anatomy and Bioimaging, Department of Cellular Function, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi 1-757, Niigata 951-8510, Japan; * [email protected] 2Johetu City Aquarium, Nishihoncho 4-19-27, Johetsu, Niigata 942-0004, Japan

Abstract: The ovary and associated appendages of the rare epipelagic octopus, Ocythoe tuberculata caught alive near the harbor of Naoetsu Port, Sea of Japan on 15 February 2005, were examined histologically. The ovary was 40 g in weight, and contained approximately 60,000 eggs. The oocytes, in variously developmental stages, were attached singly to lamella- like stalks. The ovary possesed a central cystic coele, with a complex labyrinthine form in cross section．Each ovoid oocyte in the early stage had a prominent round nucleus (= germinal vesicle) containing several nucleoli. Numerous oocytes were surrounded by an intricately folded follicular epithelial layer. Large spherical oocytes had significant yolk accumulation. Deeply infolded mucosae were present in the proximal part of the oviduct, whereas shallowly intricate mucosae were detected in the distal part of the oviduct, which was surrounded by thick muscular and fibrous connective tissues. The shape of the oocytes and pattern of oogenesis of this epipelagic octopus were similar to those of coastal and deep-sea octopods.

Keywords: ovarian histology, egg maturation, epipelagic octopus, Ocythoe tuberculata, Niigata Prefecture, Sea of Japan

Introduction

The rare epipelagic octopus, Ocythoe tuberculata Rafinesque, is widely distributed in the warm waters of the Pacific, Indian and Atlantic Oceans, the Mediterranean Sea and the Sea of Japan (Roper & Sweeney, 1975). During one winter (22 November, 2004-19 February, 2005) along the beaches of Niigata Prefecture on the west coast of Honshu (Sea of Japan), 34 stranded or drifting individuals of this species were collected by local inhabitants (Honma et al., 2005). One individual octopus, caught alive on 15 February, 2005, near the harbor of Naoetsu Port, was immediately transported to the Joetsu City Aquarium, where it died on 18 February. Because there have been no published reports on the ovarian histology of Ocythoe tuberculata, the present examination was undertaken.

Materials and Methods

Shortly after its death in the aquarium, early in the morning on 18 February 2005, an adult female Ocythoe tuberculata was dissected, and the ovary and associated appendages were removed and preserved in 10% formalin. These materials were forwarded to the Anatomy Department, Niigata University Graduate School of Medical and Dental Sciences, where the materials were refixed in Bouin’s solution to improve their staining properties. Blocks cut from several portions 346 Y. Honma, T. Ushiki, M. Takeda & Y. Nakamura were dehydrated through an alcohol series, embedded in paraffin, cut at 5 µm thickness, stained with hematoxylin-eosin (HE) double stain and Masson-Goldner (MG) associated with aldehyde fuchsin (AF) tetrachrome, and observed under the light microscope (Leitz Orthoplan). The developmental stages in the eggs, the elaboration of the follicular epithelium and the proc- ess of vitellogenesis (yolk globules and platelets) were diagnosed according to the classification of Boyle & Chevis (1992). Further, the gonosomatic index (GSI = ovarian weight/body weight ×100) was estimated.

Observations

Measurements of the specimen were as follows: total length 53.0 cm, mantle length 19.0 cm, body weight 1.5 kg, ovary weight 40 g, GSI 2.67, eggs primarily long and ovoid, of 0.5-1.0 mm in length (maximum diameter 3 mm). The eggs numbered ca. 60,000 (by visual approximation: ca. 1,500/g). The progress of development of eggs was tentatively determined and summarized as follows: Stage I. Oogonia, not yet surrounded by follicular epithelium. Stage II. Oocyte, pre-vitello- genesis. 1) Round oocyte surrounded by flat follicular epithelium. 2) Ovoid oocyte surrounded by cuboidal follicule-cells. 3) Ovoid oocyte surrounded by columnar follicle-cells. StageIII. 4) Ovoid oocyte showing early phase of follicular epithelium invagination. 5) Long ovoid oocyte with deeply invaginated follicular epithelium. 6) Oocyte with deeply invaginated follicular epithelium and thecal cells. 7) Oocyte with numerous yolk globules and distinct oolemma. 8) Oocyte with yolk platelets and amorphous-structured follicular epithelium. The ovary and oviduct, constituting a spherical mass, were surrounded by a fibrous capsule, and positioned on the liver and renal organs. The eggs (in various developmental stages) were partitioned into numerous cysts, each egg being attached singly to a lamella-like stalk (or sheath) derived from a fibrous capsule (Fig. 1). The ovary possessed oocytes in various developmental stages, showing a complex labyrinthine form around a central cystic coele in cross section. At the earliest stage, the oogonium was round in shape with no follicular epithelium (St. I). Mitotic division was only rarely detected in the oogonia gathered in the cysts (Figs. 1, 2). At the next stage, early oocytes became ovoid in shape, surrounded by a flat follicular epithelium (St. II-1). Early oocytes, 84-132 µm in diameter, had a prominent nucleus, about 36 µm in diameter. However, further development of oocytes involved very complex morphological phases. Ovoid oocytes, 15-40 µm in longer axis，had a round apically localized nucleus, with prominent nucleoli. This type of oocyte was surrounded by thin thecal and cuboidal follicular epithelial cells. Its cyto- plasm was relatively smooth and compact (St. II-2). The oocyte was then surrounded by a columnar follicular epithelium (St. II-3) (Figs. 2, 3). With further oocyte development, the folding of the follicular epithelium gradually developed from a shallow depression to several longitudinally invaginated structures (St. III-4) (Fig. 3). The size of the oocytes (follicles) measured 360-960 µm wide and 720-1200 µm long. These oocytes were surrounded by a columnar follicular epithelium (St. III-5). The epithelial cells, ranging from low to high columnar in shape, comprised pale cytoplasm and orangenophil nuclei following MG- AF staining.The outer surface of each follicle was surrounded by a single layer of thecal cells and sporadically distributed capillaries (St. III-6) (Fig. 4). An inner oolemma (= chorion) in the fol- licular epithelium was stained red with HE and positive to AF, revealing a pattern of zona radiata (Fig. 5). The ooplasm was characterized by a wavy margin forming vesicles and vacuoles. The yolk globules developed in the ooplasm in several following stages (St. III-7) (Fig. 5). The globules changed gradually into large yolk spherules, and finally formed yolk platelets. The development of yolk platelets was accompanied by a gradual decrease in the degree of follicular epithelial folding. Each mature oocyte, consisting only of yolk platelets, was surrounded by an amorphously struc- Ovarian Histology of Ocythoe tuberculata 347

Fig. 1. Section of ovary of Ocythoe tuberculata comprising oocytes in various developmental stages. Young oocytes are attached solitarily to the stalk (arrow) of the ovarian lamella. In several oocytes, prominent longitudinal enfoldings of follicular epithelia are apparent (asterisk). HE stain. Scale bar = 50 µm. Fig. 2. Massed oogonia (asterisk), a young oocyte with a nucleus (germinal vesicle) and nucleoli (arrow), and part of a more developed oocyte showing the strong invagination of follicular epithelium consisting of high columnar cells (arrow head). HE stain. Scale bar = 25 µm. Fig. 3. An oocyte showing an early phase of invagination of the follicular epithelium (asterisk), and a young oocyte comprising a round nucleus and prominent nucleoli (arrow). HE Stain. Scale bar = 25 µm. 348 Y. Honma, T. Ushiki, M. Takeda & Y. Nakamura

Fig. 4. An oocyte showing deep invgination of the follicular epithelium (asterisk) surrounded by thecal cells (arrow). HE stain. Scale bar = 25 µm. Fig. 5. Part of the mature ovum comprising numerous yolk globules (large asterisk). Ovum is surrounded in turn by: oolemma (chorion or zona radiata) (arrow head), follicular epithelium (small asterisk) and theca (arrow). HE stain. Scale bar = 25 µm. Fig. 6. Part of the mature ovum showing yolk platelets in the ooplasm. MG-AF stain. Oolemma strongly reacts with AF (arrow). Scale bar = 25 µm. Ovarian Histology of Ocythoe tuberculata 349

Fig. 7. Section of proximal oviduct showing highly intricate mucosae (arrow). HE stain. Scale bar = 50 µm. Fig. 8. Section of distal oviduct surrounded by thick muscular wall (asterisk). Note low mucosal epithelium (arrow). HE stain. Scale bar = 50 µm. Fig. 9. Part of mucosae of proximal oviduct consisting of high columnar epithelial cells (arrow heads) with basally shifted nucleus. HE stain. Scale bar = 25 µm. Fig. 10. Part of mucosae of distal oviduct consisting of low columnar epithelial cells (arrow head). HE stain. Scale bar = 25 µm. 350 Y. Honma, T. Ushiki, M. Takeda & Y. Nakamura tured follicular epithelium, consisting only of yolk platelets (St. III-8) (Fig. 6). Atretic follicles in various stages were often found. In the final stage, both thecal and chori- onal elements were almost entirely lost. In addition, no follicular cell elements were recognized. Residual ooplasm was recognized only as fragmental yolk platelets (Fig. 6). According to the above description of oogenesis, the developmental type of the eggs was determined to be synchronous. The long and winding oviduct was divided into proximal and distal portions (Figs. 7, 8). The proximal oviduct showed irregularly folded mucosae in cross section, which consisted of high columnar cells and invading submucosae. Each mucosal cell had a finely stippled nucleus located basally (Fig. 9). A wide lumen occupied the central part of the oviduct (Fig. 7), and its mucosal margin was lined with longitudinal and circular muscle layers with loose intervening connective tissue (Fig. 7). Released vacuoles were observed in the cell apices (Fig. 9). The distal part of the oviduct comprised an irregularly shaped duct in the center, and was surrounded by a very thick muscular wall, in association with fibrous connective tissue (Fig. 8). However, the degree of mucosal folding in the distal part was less developed and mucosal epithelia were lower than those in the proximal portion (Fig. 10).

Discussion

The epipelagic octopus, Ocythoe tuberculata, dwells and reproduces in surface-oceanic waters worldwide (Roper & Sweeney, 1975), being the only known cephalopod with a swim-bladder (Packard & Wurtz, 1994). Males, characterized by possessing a copulation arm termed “hectocoty- lus” (Naef, 1923; Young, 1972), are dwarf and reside, along with smaller females, within the test of the large salpa Tethys vagina (Jatta, 1896; Naef, 1923; Hardwich, 1970; Okutani & Osuga, 1986). In Japan, however, occurrences of this tuberculate octopus are rather rare. Reports of the species off the west coast of Honshu and northern Kyushu include Tanaka (1950; San-in District), Honma & Kitami (1978; Sado Island), and Honma et al. (1983; Niigata and Sado Island). A recent mass stranding along the coast of western and central areas of Niigata District during winter in 2004- 2005, was noted along with the oceanographic conditions at that time, by Honma et al. (2005). In spite of its unique form and life history, histological examinations of the ovary and oviducts of O. tuberculata have not been previously conducted. However, the histological pictures revealed by the present examination were similar to those of other cephalopod species, for example, the Chilean coast Octopus mimus (Olivares et al., 2001; Zamora & Olivares, 2004), world-wide coastal O. vulgaris, collected from the Mediterranean Sea (Di Cosmo et al., 2001) and Atlantic coast of Andalusia (Rodriguez-Rua et al., 2005), North Sea Eledone cirrhosa (Boyle & Chevis, 1991, 1992), deep sea Opisthoteuthis grimaldii, caught off west Scotland (Boyle & Daly, 2000), the com- mon flying squid, Todarodes pacificus (Takahashi & Yahata, 1974), and even the coastal cuttlefish, Sepia officinalis, and squid, Loligo vulgaris (Yung, 1930; Dhainaut & Richard, 1976). Takahashi & Yahata (1974) further noted that the reproductive mode of ovarian eggs of Todarodes pacificus was asynchronous with multiple spawning, as ascertained for the present specimen. One of the most characteristic aspects in the developmental stages of cephalopods is the pres- ence of the deep and intricately folded follicular epithelial layer (Dhainaut & Richard, 1976; Bolongnari et al., 1976; Takahashi, 1978; Boyle & Chevis, 1992; Boyle & Daly, 2000; see also Figs. 3-5, this paper). The follicular epithelial cells apparently produce a proteinous substance (Takahashi, 1978). The oocyte structure, particularly the zona pellucida, follicular epithelium, theca and sur- rounding capillaries, is comparable to that of teleosts (Bottke, 1974; Dhainaut & Richar, 1976). Generally, the histological features of the Ocythoe tuberculata oviduct were similar to those of Octopus vulgaris (Peterson, 1959; Di Cosmo et al., 2001). The only clearcut difference is in the grades of irregularity and enfolding of mucosae between the proximal and distal oviducts. As was Ovarian Histology of Ocythoe tuberculata 351 described in O. vulgaris by Di Cosmo et al. (2001), the proximal oviduct of the present species consisted of taller and more folded epithelial mucosal cells, and, the thickness of the surrounding wall was greater in the distal oviduct than the proximal oviduct. Although the epipelagic Ocythoe tuberculata is known to be ovoviviparous (Naef, 1923; Laptikhovsky & Salman, 2003), the histological architecture of its ovary is rather similar to that of coastal Octopus spp., which deposit eggs in capsules and/or jelly-like envelopes, on hard substrata (Di Cosmo et al., 2001; Rocha et al., 2001). The reproductive strategies of O. tuberculata and other cephalopods were recently reported by Rocha et al. (2001) and Laptikhovsky & Salman (2003). Oocyte size in O. tuberculata was variable according to the developmental stages; 0.2 × 0.9 mm (Naef, 1928), 1.00 × 1.75 mm (Roper & Sweeney, 1975), and 1.3-1.7 × 2.5-2.7 mm in freshly fer- tilized eggs (Laptikhovsky & Salman, 2003). The estimated size of mature eggs of O. tuberculata was variable, 1.0-3.0mm in (maximum) diameter. Laptikhovsky & Salman (2003) estimated the total fecundity of the same species as about 200,000 in one specimen of 170 mm in mantle length, which had some 170,000 ovarian oocytes at different stages of development. Naef (1923) reported at least 100,000 eggs kept in the oviducts of a mature female. By comparison, we estimated about 60,000 oocytes in various developmental stages in the present 190 mm mantle length specimen of O. tuberculata.

References

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(Received November 30, 2005 / Accepted April 27, 2006) Ovarian Histology of Ocythoe tuberculata 353

新潟県直江津港沖で採捕された浮遊遠洋性アミダコ卵巣の組織像 ― 海産無脊椎動物の生殖腺の成熟に関する研究－XIII ―

本間義治・牛木辰男・武田正衛・中村幸弘

要 旨

浮遊遠洋性の珍蛸アミダコ（♀）が，2004 年 11 月から 2005 年 2 月下旬にかけて，上・中越地方を中 心に新潟県沿岸へ漂着したり捕獲されたりして，34 個体が記録された．これらの中で，2 月 15 日に掬わ れ，上越水族博物館へ収容され，18 日に死亡した全長 53 cm の個体を 10 ％フォルマリンで，次いでブ アン氏液で固定し，卵巣・卵管の組織標本を作成して，観察した．卵巣重量は 40 g，抱卵数は 60,000 個 以上であった．卵巣は中央に卵巣腔があり，多数の包嚢からなり，嚢内には様々の発育段階の卵巣卵（非 同時発生型）が存在していた．若い卵母細胞は，それぞれ結合組織性の薄膜（層板）に付着していた． 初期の卵母細胞には，円形の核（生殖胞）が明瞭であるが，発育が進むと卵は長楕円形となり，卵胞上 皮が随所から陥入し始め，複雑に入り組み，卵黄形成が盛んとなる．さらに成熟が進むと，卵母細胞は 大きく球状化して，最大径 1.2 mm に達する．卵膜には放射線帯，卵胞膜，莢膜の分化が明瞭となり，卵 黄は板状化する．近位卵管内壁の粘膜は高く，複雑にひだ打っているが，遠位卵管壁は厚い結合組織と 筋肉層で覆われ，内壁の粘膜は低く，ひだ打ちの程度は小さかった．浮遊遠洋性アミダコ卵母細胞の成 熟過程は，沿岸性のマダコや深海性のメンダコ類などと変わらず，多回産卵を行うと推定された．