Blackwell Science, LtdOxford, UK FISFisheries Science0919-92682003 Blackwell Science Asia Pty Ltd 695October 2003 707 Reproductive cycle of black rockfish H Mori et al. 10.1046/j.0919-9268.2003.00707.x Original Article910923BEES SGML FISHERIES SCIENCE 2003; 69: 910–923 Annual reproductive cycle of black rockfish Sebastes schlegeli in captivity Hisanari MORI,1 Masahiro NAKAGAWA,2 Kiyoshi SOYANO3 AND Yasunori KOYA1* 1Department of Biology, Faculty of Education, Gifu University, Gifu, Gifu 501-1193, 2Miyako Station, Japan Sea-Farming Association, Miyako, Iwate 027-0097 and 3Department of Biology, Faculty of Fisheries, Nagasaki University, Nagasaki, Nagasaki 851-2213, Japan ABSTRACT: The gonadal development and serum profiles of vitellogenin and sex steroids in rearing Sebastes schlegeli were monitored for one full year. Female fish began vitellogenesis from November and completed it in March. Gestation occurred from April, and parturition occurred in June. A thin chorion and scanty cortical alveoli are oogenetic peculiarities of this fish. Male fish began spermatogenesis from June, and matured in November and December. It appears that copulation occurs in November and December, and that the sperm are stored freely in the ovary during the early vitellogenic period and under the ovigerous lamellae epithelium during the late vitellogenic period. Serum vitellogenin levels in female fish had a good correlation with oocyte growth. Serum estradiol- 17b (E2) levels in female fish were elevated from November to February, suggesting that E2 controls vitellogenesis. Serum 17,20b-dihydroxy-4-pregnen-3-one (DHP) in female fish increased in the late vitellogenic period, suggesting that DHP was a maturation-inducing steroid. High levels of serum DHP during gestation suggest that it may be one of the endocrine factors for maintaining gestation. Serum 11-ketotestosterone (11-KT) levels in male fish were elevated from June to October, suggest- ing that 11-KT controls spermatogenesis. Serum DHP in male fish had a single peak in October, suggesting that DHP plays some role in the late stages of spermatogenesis. KEY WORDS: oogenesis, reproductive cycle, sex steroids, spermatogenesis, sperm storage, vitellogenin, viviparous teleost. INTRODUCTION important roles in oocyte growth (vitellogenesis) and final oocyte maturation. Vitellogenin (Vg), a Black rockfish Sebastes schlegeli is a viviparous yolk-precursor protein, is synthesized in the liver teleost belonging to the Scorpaenidae and inhab- by estrogen stimulus and incorporated into grow- iting the coast of Japan, Korea, and China. In the ing oocytes, then stored as yolk globules. The full- Scorpaenidae, 330 species have been confirmed, grown oocytes undergo final maturation under 110 of which are viviparous. Many species are uti- the stimulus of a maturation-inducing steroid lized as fisheries resources, especially black rock- (MIS). Some studies on vitellogenesis in the vivip- fish, which is an important species for artificial arous rockfish have been carried out on the basis seedling production and juvenile release. Many of histological observations of the ovary,2–4 and the institutes are engaged in the mass production of serum profiles of Vg and estrogen.5–7 The results of juveniles, and much information on the artificial these studies confirm that the hormonal regula- production of juveniles in this fish has accummu- tion of vitellogenesis is the same as that in ovipa- lated.1 However, the studies on the reproductive rous teleosts. In contrast, studies of oocyte final physiology of this species are limited and fragmen- maturation in viviparous teleost are limited only tary. In order to develop efficient techniques for the to white-edged rockfish, S. taczanowskii, on the artificial production of juveniles, basic information basis of in vitro experiments of the induction of on reproductive physiology is necessary. oocyte maturation and production of hormones in As for the reproductive physiology of female the ovarian follicle by gonadotropin (GTH) stimu- teleosts, it is well known that some hormones play lation,8 together with the serum profiles of some steroid hormones.7 In white-edged rockfish as well 9,10 *Corresponding author: Tel: 81-58-293-2255. as other oviparous species, it is suggested that Fax: 81-58-293-2259. Email: [email protected] the MIS is 17,20b-dihydroxy-4-pregnen-3-one 8 Received 18 November 2002. Accepted 3 April 2003. (DHP). Reproductive cycle of black rockfish FISHERIES SCIENCE 911 Androgens play important roles in male repro- To identify cortical alveoli in the oocytes, some duction in fish, including testicular development. sections were treated with periodic acid–Schiff 11-ketotestosterone (11-KT) is detected in the (PAS) reagent. To identify oil droplets in the maturing male fish of many teleost species, and oocytes, a part of each ovary was prefixed in a glu- is accepted as a major androgen inducing sper- taraldehyde (2.5%)–paraformaldehyde (4%) mix- matogenesis.11,12 After spermatogenesis the testes ture in a 0.2 M cacodylate buffer (pH 7.4). After change from synthesizing mainly androgens to fixation for 5 h at room temperature the specimens mostly progestins in many male teleosts.13 The were washed three times with 0.1 M cacodylate progestins induce spermiation and are respon- buffer (pH 7.4) for 12 h at 4∞C, postfixed at 4∞C for sible for sperm motility.14 In some teleosts DHP 2 h in 1% osmium tetroxide in a 0.1 M cacodylate has been numbered among the main male buffer, dehydrated through a graded alcohol series, progestins.13–16 Although some histological studies and embedded in epoxy resin. Semi-thin sections of the testes of viviparous rockfish have been car- were cut and stained with a 1% solution of tolui- ried out,17–20 there are no reports on the changes in dine blue. blood concentrations of some steroid hormones in To measure oocyte follicle diameters, a part of male rockfish. each ovary was preserved in 10% formalin. The In the present study we monitored the ovarian diameters of 20 developing oocytes in each female and testicular development as well as serum pro- fish were measured using a projector. files of Vg and sex steroid hormones in the black The microscopic images (¥400) randomly cho- rockfish for an entire year, using rearing fish in sen from three fields within each cross-section of order to clarify the peculiarities of gametogenesis the testis were captured by video. A sheet of tracing and its hormonal regulation in viviparous species. paper was attached to the monitor for each image. The areas occupied by the different spermatogenic cells or cysts (e.g. spermatogonia, spermatocytes, MATERIALS AND METHODS spermatids and spermatozoa) were traced, cut and weighed. The percentage of each type was Fish and samples expressed as the ratio of the area occupied by a particular spermatogenic cell stage to the total area Seven to nine-year-old black rockfish used in the of all spermatogenic cells. The presence of sperma- present study were produced in the aquaria of the tozoa in the sperm duct was also examined in each Japan Sea–Farming Association, Miyako Station, fish. Iwate Prefecture, Japan. Five male and five female fish were sampled every month between Decem- ber 1997 and November 1998. After the fish were Measurement of vitellogenin levels measured for standard length and body weight, they were bled from the caudal vessel with non- Black rockfish serum Vg levels were measured heparinized syringes. Sera were separated by cen- by an immunological assay using an antiserum trifugation at 3000 ¥g for 15 min and stored at - against purified Vg. The Vg was induced by 80∞C until use. The testes or ovaries were dissected estradiol-17b (E2)-injected male fish (BW: 0.87– and weighed. The gonadosomatic index (GSI) was 1.37 kg) in May. A solution of E2 was made by dis- calculated as follows: GSI = GW ¥ 100/BW, where solving it in propylene glycol at a concentration of GW and BW are gonadal weight (g) and body 10 mg/mL. Male fish received intramuscular injec- weight (g), respectively. The liver weight (LW; g) tions of E2 solution three times at 2 day intervals at was measured, and the hepatosomatic index (HSI) a dose of 20 mg per kg of body weight. Fish were was calculated as follows: HSI = LW ¥ 100/BW. bled from the caudal vein 2 days after the third injection. The E2-treated male serum (E2S) was separated by centrifugation by the same method Observation of gonads described earlier, and stored at -80∞C until use. The E2S was applied to a hydroxylapatite (Bio- Ovarian fluid was collected by cannulation from Rad Laboratories, Hercules, CA, USA) column genital pores and examined under light micro- (ø2.5 ¥ 3.0 cm) equilibrated with 0.5 M potassium scope for the presence of sperm. For histological phosphate buffer (KP; pH 7.0). After washing with observations of gonads, part of the testis or ovary the same buffer, bound proteins were dissociated was fixed in Bouin’s solution, dehydrated with eth- with 1.5 M KP (pH 7.0; elution speed: 0.6 mL/min). anol, and embedded in paraffin. The specimens The eluted fractions showing a high absorbance at were cross-sectioned at 6 mm and stained with 280 nm were concentrated with Ms. BUTAURY-KN Delafield’s hematoxylin and eosin. (Atto, Tokyo, Japan), and further applied to a 912 FISHERIES SCIENCE H Mori et al. Sephadex G-200 (Amersham Pharmacia Biotech estimated using the following formula: (ring UK, Buckinghamshire, UK) column (ø3.5 ¥ diameter)2 - (well diameter)2. The lower detection 104.0 cm) equilibrated with 20 mM Tris-HCl buffer limit was approximately 0.5 mg/mL. (pH 8.0) containing 150 mM NaCl and 0.001% NaN3. A single symmetrical peak was obtained in the chromatogram, and the peak fraction was col- Enzyme immunoassay for steroid hormones lected as purified Vg.