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