Annual Reproductive Cycle of Black Rockfish Sebastes Schlegeli In

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, suggesting 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. An emulsion of 0.5 mL of purified Vg and an Estradiol-17b, testosterone (T), 11-KT and DHP in equal volume of Freund’s complete adjuvant serum were measured by specific enzyme-linked (Wako Pure Chemical Industries, Osaka, Japan) immunosorbent assay (ELISA) according to the was injected intradermally into the back of a rabbit method of Asahina et al.23 Values under the detec- four times at weekly intervals. The rabbit was bled tion limit (50 pg/mL in all steroids) were consid- from a vein in the ear after the fourth injection. ered as 50 pg/mL for statistical analysis. Serum was separated by centrifugation at 3000 ¥g for 15 min at room temperature and stored at - 80∞C until use as rabbit antiserum against rockfish Statistics Vg (a-Vg). Specificity of a-Vg was assessed by immunoeloctrophoresis using male and female All data were presented as mean ± SEM, and were serum and vitellogenic oocyte extract. analyzed by one-way ANOVA to test for the differ- Serum Vg concentrations were measured by the ences among groups. The means were subse- method of Mancini et al.21 using the purified Vg as quently compared by Fisher’s protected least a standard. The protein concentration of Vg frac- significant difference (PLSD) test using the STAT- tions was determined by the method of Lowry VIEW 4.5 program for Macintosh (Abacus Concepts, et al.22 using bovine serum albumin as a standard. Berkeley, CA, USA). Differences were considered Antiserum (a-Vg) was diluted to 2.5% in 1% agarose statistically significant at P < 0.05. dissolved in 0.9% NaCl containing 0.01% NaN3 at 56∞C. Five microliters each of female sera and seri- ally diluted Vg fraction were applied to the wells of RESULTS the gel plate and incubated for 48 h in a moisture chamber at room temperature. After washing, dry- Annual changes in gonadosomatic and ing, and staining with Amido Black 10B (Wako Pure hepatosomatic indices Chemical Industries), the diameters of immuno- precipitation circles were measured to 0.1 mm, and The GSI values of female fish (Fig. 1a) continued to the relative concentrations of the antigen were increase from December (3.6 ± 0.3%) to a peak

(a) (c)

(b) (d)

Fig. 1 Annual changes in gonadosomatic index (GSI) and hepatosomatic index (HSI) of female and male black rockﬁsh. (a) female GSI; (b) female HSI; (c) male GSI; (d) male HSI. *Signiﬁ- cantly different from each other. n = 5 in each sampling.

Reproductive cycle of black rockﬁsh FISHERIES SCIENCE 913

level (42.5 ± 4.1) in May. They then decreased dra- ooplasm. The oil droplets become distributed over matically in June (1.8 ± 0.1%), and maintained a the cytoplasm. The PAS-positive granules are low level (1.1–1.8%) from July to November. The still found. The chorion is approximately 2 mm in HSI values of female fish (Fig. 1b) remained high thickness. (2.2–2.4%) from December to March, but then (7) Tertiary yolk globule stage: oocytes are decreased in April (1.6 ± 0.3%), exhibiting a mini- 590–950 mm in diameter (Fig. 2h). Yolk globules mum level (1.2 ± 0.1%) in May. The HSI values then continue to accumulate in the ooplasm. The PAS- increased in June, and held steady at 1.3–1.9% from positive granules are no longer seen. In the semi- July to November. thin epoxy section stained with toluidine blue, The GSI values of male fish (Fig. 1c) decreased cortical alveoli-like structures (7–10 mm in diame- from December (0.59 ± 0.09%) to January ter) are distinguishable from yolk globules in the (0.30 ± 0.02%), and remained low until August. cortical cytoplasm. The chorion is approximately They rapidly increased from August to a peak 3.5 mm in thickness. (1.44 ± 0.06) in October, and maintained a high (8) Migratory nucleus stage: oocytes are 920– level in November (1.43 ± 0.08%). The HSI values 1040 mm in diameter (Fig. 2i). The germinal vesicle of male fish (Fig. 1d) continued to rise from migrates to the cortical region of the ooplasm. The December (1.5 ± 0.12%) to a peak level in May chorion is still approximately 3.5 mm in thickness. (4.3 ± 0.42%), then continued to decrease to the The micropyle is observed in the chorion of the lowest level in November (1.4 ± 0.12%). animal pole region (Fig. 2j). The chorion of the micropylar region is 7.4 mm in thickness, which is thicker than in the other regions. The micropylar Seasonal changes in ovaries vestibule is approximately 4.5 mm in diameter, and the micropylar tube narrows toward the ooplasm. Oocytes of black rockfish were divided into eight The oocytes at each developmental stage in the developmental stages, following the corres- ovary and the presence of the embryos were exam- ponding division of white-edged rockfish, ined (Table 1). Oocytes at the chromatin nucleolus S. taczanowskii.4 stage were excluded because they were too small (1) Chromatin nucleolus stage: oocytes approx- to count accurately. Oocytes at the early and late imately 13 mm in diameter have a spherical nucleus peri-nucleolus stages were observed throughout with a large chromatin nucleolus occupying a the year in all fish. In December, all fish had pri- major part of the oocyte. mary and secondary yolk globule stage oocytes, (2) Early peri-nucleolus stage: oocytes 30–60 mm and three out of five fish had oil droplet stage in diameter have ooplasm strongly stained with oocytes including vitellogenic oocytes. In January hematoxylin (Fig. 2a). Several nucleoli are distrib- all female fish had oocytes at the secondary yolk uted along the nuclear membrane. globule stage. In February, all female fish had (3) Late peri-nucleolus stage: oocytes 50– oocytes at the tertiary yolk globule stage. In March, 130 mm in diameter show increased volumes of oocytes at the migratory-nucleus stage appeared both ooplasm and nucleus (Fig. 2b). The ooplasm in three of five fish. In April, embryos were con- becomes slightly less reactive to hematoxylin com- firmed in three of four fish, indicating that preg- pared with the former stage. nancy had started. One fish had oocytes at both (4) Oil droplet stage: oocytes are 110–270 mm in the migratory nucleus and tertiary yolk globule diameter. The oil droplets apparent in the ooplasm stages at this time. In May, vitellogenic oocytes showed vacuoles in the paraffin section (Fig. 2c) were no longer observed, and growing embryos and are stained weakly brown in the osmium-fixed were found in the ovaries of all fish. In June, no epoxy section (Fig. 2d). embryos were observed, indicating that parturi- (5) Primary yolk globule stage: oocytes are 260– tion was complete. The leading oocyte stage was 450 mm in diameter (Fig. 2e,f ). Yolk globules the oil droplet stage in most of the fish. From stained with eosin begin to appear in the periph- August to October, the ovaries of all fish contained eral ooplasm. Oil droplets of various sizes are non-vitellogenic oocytes. In November, oocytes at found in cytoplasm surrounding the nucleus. The the primary yolk globule stage appeared in the PAS-positive granules thought to be cortical alveoli ovaries of all fish. (yolk vesicles) are present in the cytoplasm Monthly changes in the average diameter of the (Fig. 2f ). The chorion (zona radiata) is approxi- major ovarian follicle in every individual were mately 1 mm in thickness, and is PAS-positive. examined (Fig. 3). In December, ovarian follicles (6) Secondary yolk globule stage: oocytes are were 0.52 ± 0.03 mm in diameter, increasing lin- 420–610 mm in diameter (Fig. 2g). The yolk globules early until March (1.25 ± 0.03 mm), and reaching a continue to increase in number and fill the entire peak in April (1.36 ± 0.02 mm). In May all fish had

914 FISHERIES SCIENCE H Mori et al.

Fig. 2 Histological observations of oocytes in black rockfish.(a) early peri-nucleolus stage (bar = 10 mm); (b) late peri- nucleolus stage (bar = 20 mm); (c) oil droplet stage (bar = 50 mm); (d) oil droplet stage (bar = 50 mm). Epoxy section with toluidine blue staining; (e) primary yolk globule stage (bar = 50 mm); (f) primary yolk globule stage (bar = 50 mm). Periodic acid–Schiff (PAS) staining; (g) secondary yolk globule stage (bar = 100 mm); (h) tertiary yolk globule stage (bar = 200 mm); (i) migratory nucleus stage (bar = 200 mm). Arrow indicates nucleus; (j) micropyle apparatus in migratory nucleus stage oocyte (bar = 5 mm); (k) sperm (arrowheads) in the ovary in February. Arrow indicates chorion (bar = 5 mm). become pregnant, and there were no developing ming sperm were observed in them. However, oocytes. From June to September the ovarian sperm could not be identified from the histological follicles were too small to distinguish. In October, sections in December. In contrast, although sperm ovarian follicles 0.26 ± 0.01 mm in diameter were were not observed in the ovarian fluid from Janu- distinguished, increasing to 0.37 ± 0.02 mm in ary to March, many were identified in the histolog- November ical sections during those months (Fig. 2k). The In both November (just before the first sam- stored sperm seem to have penetrated beneath the pling) and December, small amounts of ovarian ovigerous lamellae epithelium that covered each fluid were collected from genital pores, and swim- ovarian follicle. Reproductive cycle of black rockfish FISHERIES SCIENCE 915

Table 1 Monthly changes of each stage of oocytes and embryos in black rockfsh† Stage Month EPN LPN OD PYG SYG TYG MN EMB Dec + +++ + + +++ + + +++ + ++ ++ ++ ++ Jan ++ ++ ++ ++ ++ ++ ++ + ++ + Feb ++ ++ ++ ++ ++ + ++ + ++ + Mar ++ + ++ + ++ ++ ++ ++ ++ ++ Apr ++ ++ ++ + ++ + ++ + May ++ + ++ + ++ + ++ + ++ + Jun ++ +++ +++ +++ +++ Aug +++ +++ +++ +++ +++ Oct +++ +++ +++ +++ +++ Nov + +++ + +++ + +++ + +++ + +++

† Existence in each ovary indicated by mark +. EPN, early peri-nucleolus stage; LPN, late peri-nucleolus stage; OD, oil droplet stage; PYG, primary yolk globule stage; SYG, secondary yolk globule stage; TYG, tertiary yolk globule stage; MN, migratory nucleus stage; EMB, developing embryos. 916 FISHERIES SCIENCE H Mori et al.

Seasonal changes in testicular histology

The relative abundance of spermatogenic cells in the testis is shown in Table 2, and histological images of testes are shown in Fig. 4. The percentage of spermatogonia was very low in December (Fig. 4e) but gradually increased from January to a peak level of 72% in April (Fig. 4b), then decreased rapidly to June (Fig. 4c). From July to November, the spermatogonia cysts could not be detected in three randomly chosen fields of sections, but the cysts of spermatocytes and spermatids were fre- quently observed (August; Fig. 4d). The percentage of spermatocytes gradually increased from April (Fig. 4b) to a peak level of 78% in June (Fig. 4c), and then gradually decreased to November. The percentage of spermatids changed at low levels (0– Fig. 3 Annual changes in oocyte follicle diameters in 33%) compared with that of other spermatogenic black rockfish. Every value is significantly different. n = 5 cells, with their highest level observed in January. in each sampling. The percentage of spermatozoa was high (79%) in

Fig. 4 Histological observations of testis in black rockfish. g, spermatogonia; c, spermatocytes; t, spermatids; z, spermatozoa. (a) Recovery period (March); (b) spermatogonial proliferation period (April); (c) early spermatogenic period (June); (d) late spermatogenic period (August); (e) functional maturation period (December; bar = 100 mm; same magnification for a–e); (f) sperm duct in October; bar = 500 mm. Reproductive cycle of black rockfish FISHERIES SCIENCE 917

Table 2 Monthly changes in frequency of each stage of germ cells in the testis and appearance of sperm in the sperm duct in male black rockﬁsh Stage Month n SG SC ST SZ SD† Dec5 – 9.8 ± 1.78 11.2 ± 2.13 79.0 ± 3.30 5 Jan50.4 ± 0.26 17.6 ± 3.68 32.9 ± 12.34 49.1 ± 14.75 5 Feb515.3 ± 15.30 51.3 ± 13.41 22.6 ± 6.55 10.8 ± 6.06 5 Mar557.8 ± 1.48 42.2 ± 1.48 – – 5 Apr571.7 ± 7.09 28.3 ± 7.08 – – 2 May559.2 ± 11.57 40.8 ± 11.57 – – 0 Jun50.5 ± 0.33 77.9 ± 9.62 16.6 ± 6.77 5.0 ± 4.16 0 Jul5 – 66.6 ± 5.92 16.1 ± 4.27 17.3 ± 2.70 3 Aug5 – 35.7 ± 3.47 18.5 ± 3.94 45.8 ± 5.74 4 Oct5 – 30.1 ± 30.16 13.8 ± 4.49 56.1 ± 2.57 5 Nov 5– 7.7 ± 1.18 6.6 ± 1.17 85.7 ± 2.07 5

SG, spermatogonia; SC, spermatocytes; ST, spermatids; SZ, spermatozoa. Mean ± SEM. † Numerals indicate the number of ﬁsh which spermatozoa were observed in the sperm duct.

rapidly in May and maintained low levels of 1.45– 0.64 mg/mL until October, increasing to 7.6 ± 1.5 mg/mL again in November.

Seasonal changes in serum steroid hormone levels

Serum E2 levels of female fish (Fig. 6a) increased from December (12.8 ± 0.3 ng/mL) to a peak level in February (29.6 ± 3.0 ng/mL). They then decreased until April (2.8 ± 1.1 ng/mL) and Fig. 5 Annual changes in serum vitellogenin levels in were maintained at a low level until October female black rockfish. *Significantly different from each (2.9 ± 0.4 ng/mL). In November they increased other. n = 5 in each sampling. again to 13.0 ± 2.0 ng/mL. Serum T levels of female fish (Fig. 6b) increased from December (2.5 ± 0.4 ng/mL) to February December (Fig. 4e) but rapidly decreased to zero in (7.4 ± 1.7 ng/mL), were maintained at a high March (Fig. 4a). In June the spermatozoa reap- level until April, decreased in May, and stayed peared, and gradually increased to a peak level of at low levels (0.4–1.8 ng/mL) between June and 86% by November. November. Spermatozoa were observed in the sperm duct Serum DHP levels of female fish (Fig. 6c) were in all samples from October (Fig. 4f) to March, and low from December to January (0.3–0.4 ng/mL) in some samples in April, July and August (Table 2). and increased rapidly from February to April. They By May and June, no spermatozoa were present in remained high between April and May (7.2–7.4 ng/ the sperm duct. mL) but then increased rapidly in June, remaining at low levels from July to November. Serum 11-KT levels of male fish (Fig. 7a) Seasonal changes in female serum decreased from December (2.8 ± 1.4 ng/mL) to vitellogenin levels January (0.2 ± 0.2 ng/mL), and then stayed low until June. They gradually increased until August Seasonal changes in the serum Vg levels of female (2.9 ± 0.6 ng/mL), rapidly increased to a peak fish are shown in Fig. 5. Those levels increased (13.4 ± 1.9 ng/mL) in October, and remained high from December (14.5 ± 1.8 mg/mL) to a peak level until November. in February (23.9 ± 1.8 mg/mL), then temporarily Serum T levels of male fish (Fig. 7b) decreased decreased to 18.2 ± 3.5 mg/mL in March, but from December (1.0 ± 0.3 ng/mL) to January increased again in April. They then decreased (0.4 ± 0.1 ng/mL), and stayed low until August. 918 FISHERIES SCIENCE H Mori et al.

(a) (a)

(b) (b)

Fig. 7 Annual changes in serum steroid hormone levels Fig. 6 Annual changes in serum steroid hormone levels in male black rockfish. (a) testosterone; (b) 11-ketotest- in female black rockfish. (a) estradiol-17b; (b) testoster- osterone; (c) 17,20b-dihydroxy-4-pregnen-3-one. *Sig- one; (c) 17,20b-dihydroxy-4-pregnen-3-one. *Signifi- nificantly different from each other. n = 5 in each cantly different from each other. n = 5 in each sampling. sampling. ND, not detected. ND, not detected.

diameter. The GSI values further increased from They increased rapidly to a peak (3.3 ± 0.4 ng/mL) April to May when female fish were pregnant, in October, then decreased significantly but still and decreased rapidly by parturition in June, main- showed high levels (2.2 ± 0.3 ng/mL) in November. taining low levels until October when Serum DHP levels of male fish (Fig. 7c) vitellogenesis had not yet started. Thus, the decreased significantly from December (0.30 ± changes in the GSI values of female fish may reflect 0.04 ng/mL) to January (0.09 ± 0.04 ng/mL), and ovarian conditions. remained low until August. They then quickly The HSI values of female fish were kept rela- soared to a peak of 0.75 ± 0.13 ng/mL in October, tively high from November to March when vitello- and decreased rapidly in November. genesis was confirmed, indicating that Vg was synthesized in the liver during this period. In contrast, the HSI showed low levels from April to May, DISCUSSION when female fish were pregnant. In some viviparous teleosts the HSI values decrease during the In November when the beginning of vitellogenesis gestation period, which is thought to be due to the was confirmed by ovarian histology, the GSI values nutrition supply from mother to embryo.24–26 It was of female fish began to increase, and continued to thought that the genus Sebastes retains a primitive do so from December to March. In this period, style of viviparity known as lecithotrophy, by which vitellogenesis was confirmed by histological embryos develop to depend for nourishment on observation and by the linearly increasing oocyte their own egg yolk.27 However, recent studies Reproductive cycle of black rockfish FISHERIES SCIENCE 919

suggest that Sebastes embryos also receive some 1 month in S. inermis inhabiting warm water,2 nutrition from sources beside their egg yolk.28–30 whereas in the white-edged rockfish inhabiting Therefore, the decrease in HSI during gestation in cold water it lasts for approximately 6 months.5 the present study may be associated with the Sebastes marinus, inhabiting more northern waters nutrition supply from mothers to embryos. than the white-edged rockfish, has an even longer In the present study, oocytes at the peri- vitellogenic period (7–9 months).34 The site of the nucleolus stages were observed throughout the present experiment, Iwate Prefecture, is located year. Oocytes at the oil droplet stage appeared in between the latitudes inhabited by the white- June. They developed to the primary yolk globule edged rockfish and S. inermis. In the present study, stage from November to December, and further black rockfish needed approximately 5 months for developed as a single clutch after that. This indi- vitellogenesis, which agrees with the finding of cates that the developmental type of the oocytes in Takahashi et al.25 this species is the group-synchronous type classi- In the black rockfish, although sperm were fied by Takano.31 Cortical alveoli of black rockfish found in the ovarian fluid in November and oocytes appeared at the primary yolk globule stage. December, they could not be found in the histolog- The diameters of cortical alveoli (3–5 mm) were ical section in those months. This indicates that somewhat smaller than those of other teleost spe- copulation occurs from November to December, cies (herring, 2–25 mm;32 Fundulus, >60 mm33). The suggesting that the sperm float freely in the ovarian PAS-positive cortical alveoli disappeared in the ter- fluid in the early period of sperm storage. In con- tiary yolk-globule-stage oocyte. The PAS-positive trast, from January to March as seen on the cortical alveoli are scanty in the vitellogenic histological sections, the sperm penetrate oocytes of white-edged rockfish.4 However, in the beneath the ovigerous lamellae epithelium. In present study cortical alveoli-like structures were Sebastodes (=Sebastes) paucispinis, the sperm were found in the cortical cytoplasm of the tertiary yolk embedded among the epithelial cells.3 In white- globule stage by observation of a semi-thin epoxy edged rockfish, the sperm either adhere to the epi- section. This indicates that in viviparous rockfish, thelium of the ovigerous lamella or they are few cortical alveoli are formed, and that their con- wrapped in its microvilli.4,35 In the black rockfish, tents may change during oocyte growth. the morphology of sperm storage in the ovary may In the present study the reproductive cycle of be somewhat specialized compared with other female black rockfish was divided into the follow- Sebastes species. ing five periods. Changes in the serum Vg levels agreed well with (1) Vitellogenesis onset period (November– the development of oocytes during the vitellogenic December): oocytes at the oil droplet stage start period. After complete vitellogenesis, serum Vg yolk accumulation. Copulation occurs, and the levels remained comparatively high until April sperm are stored and swim freely in the ovarian when gestation began. In white-edged rockfish, fluid. serum Vg levels dramatically decreased during ges- (2) Vitellogenic period (January–March): oocytes tation.5 Apparent discrepancies with the present continue to accumulate egg yolk, and oocyte diam- results may be due to the differences in our respec- eter increases linearly to a maximum of 1.25 mm. tive rearing environments because serum steroid Sperm are stored under the ovigerous lamellae epi- hormone concentrations in the present study were thelium. extremely high compared with those reported (3) Gestation period (April–May): ovulated eggs previously for black rockfish and white-edged are fertilized by the sperm in the ovarian cavity, rockfish.7 and embryonic development occurs there. Ovary Serum E2 levels of female black rockfish weight continues to increase during this period increased in the onset period of vitellogenesis, due to the growth of each embryo. peaked in the vitellogenic period, and decreased by (4) Parturition period (June): embryos hatch the gestation period. These results agree with a pre- out in the ovarian cavity and are released into the vious report of annual changes in E2 concentra- seawater. tions in the same species, and suggest that E2 also (5) Resting period (June to October): some plays an important role in the vitellogenesis of oocytes at the peri-nucleolus stage develop to the viviparous Sebastes species, but not for maintaining oil droplet stage and remain there to grow until the gestation.7 However, after the serum E2 level onset of vitellogenesis. decreased from March, Vg levels still remained high Takahashi et al. noted that northern species of until May. This may have been caused by a decrease Sebastes tend to prolong gametogenesis while the in Vg uptake, because vitellogenic oocytes disap- southern species have the opposite tendency.25 peared from March. Similar phenomena were seen Vitellogenesis finishes after approximately in rainbow trout36 and Pacific herring.37 Serum T 920 FISHERIES SCIENCE H Mori et al.

levels of female black rockfish showed the same numbers increase. More than 50% of the testes is changes as those in E2 until peaking in February, occupied by spermatogonia. but the levels stayed high until April and then grad- (2) Early spermatogenic period (June–July): the ually decreased until June. Similar changes in T dur- percentage of spermatocytes rapidly increases (to ing the vitellogenic and early gestation periods were >50%), and spermatids and spermatozoa also reported in white-edged rockfish.7 It is thought that appear in the testis. the high T levels during the late vitellogenic period (3) Late spermatogenic period (August–Octo- are due to end of the conversion from T to E2 for ber): the percentage of spermatocytes decreases, this period, and that the changes in T levels reflect and that of spermatids and spermatozoa rapidly changes indicative of precursors of E2. increases, accounting for the majority of testicular Serum DHP levels of female black rockfish cysts. The GSI values rapidly increase in this rapidly increased to replace E2 in the closing period. period of vitellogenesis. These results suggest that (4) Functional maturation period (November– DHP participated in the final oocyte maturation in December): spermatozoa account for more than this species. In white-edged rockfish, in vitro 70% of the testis. The sperm duct is replete with experiments suggest that DHP is a maturation- spermatozoa. inducing steroid.8 The DHP levels stayed high not (5) Recovery period (January–February): the only during late vitellogenesis but also during ges- percentage of spermatids and spermatozoa tation in both white-edged and black rockfish.7 decreases, and spermatogonial cysts appear. Moreover, in grass rockfish, S. rastrelliger, serum Residual spermatozoa are found in the sperm duct. levels of C-21 steroids including DHP increased In S. inermis and Sebastiscus marmoratus during gestation. However, 17,20b-dihydroxy-5b- inhabiting warm water, the resting period pregnen-3-one remained the predominant steroid (during which spermatogonial proliferation lasts detected.38 In the present study DHP also stayed at <7 months), spermatogenesis occurs in a compar- high levels during gestation, suggesting the possi- atively short period of only 4 or 5 months.17,18 In bility that C-21 steroids play some role not only in contrast, in cold water or deep water species such oocyte maturation but also in gestation in the as white-edged rockfish,19 kichiji rockfish, Sebas- genus Sebastes. The site of DHP synthesis during tolobus macrochir,39 and blackbelly rosefish, Heli- gestation is thought to be postovulatory follicles colenus dactylopterus dactylopterus,40 there is no based on electron microscopic observations in resting period, and every stage of spermatogenic white-edged rockfish,34 and of in vitro experi- germ cells is observed throughout the year. The ments in black rockfish (Mori et al., unpubl. data, present results indicate that the testicular develop- 2002). ment of black rockfish belongs to the group of cold Male GSI values began to increase from August water or deep water types. to October when active spermatogenesis occurs, In many male teleosts, serum 11-KT levels possibly due to an increase in germ cell numbers increase with spermatogenesis.41–45 In black rock- by mitosis and meiosis. In November, GSI values fish, male serum 11-KT levels gradually increased stayed high, and sperm were found in the ovarian from the early spermatogenic period, rising rapidly fluid, indicating that copulation was carried out in during the late spermatogenic period, and main- this period. They decreased from December to Jan- taining high values during the functional matura- uary when reproduction (copulation) ended, sug- tion period. Although the changes in male serum T gesting that sperm release and the elimination of levels had a pattern similar to 11-KT, they were residual spermatozoa occurred during that period. lower by approximately 25%. These results strongly Thus, male GSI values accurately reflect the matu- suggest that 11-KT is the main androgen in black rity of the testes. Male HSI values showed simple rockfish, and regulates spermatogenesis in this annual changes, increasing from December to May species. The profiles of T may reflect the changes and decreasing from May to November. This pat- as precursors of 11-KT. tern of change is thought to reflect the nutritive It is known that serum DHP levels rise during condition of male fish (i.e. male fish accumulate the period of milt production in male teleo- nutritious from winter to spring and expend them sts,13,15,45–48 and that DHP induces the acquisition of from summer to autumn). Consumption of nutri- sperm motility through the elevation of pH in the ents may be due to reproductive activity. seminal plasma.14,49 However, there are no reports In the present study, the reproductive cycle of describing the relationship between DHP and male black rockfish was divided into the following spermiation in viviparous teleosts. The serum DHP five periods. levels of male black rockfish showed a single peak (1) Spermatogonial proliferation period in the late spermatogenic period (October), but (March–May): the spermatogonia divide and their rapidly declined during the functional maturation Reproductive cycle of black rockfish FISHERIES SCIENCE 921

period. This suggests that DHP may play some role Miyako Station, for their valuable assistance with in the late stage of spermatogenesis. However, fish rearing and sampling. We also wish to thank questions remain about the relation of DHP to the Mr Haruyoshi Okuya, Faculty of Education, Gifu induction of spermiation and sperm motility. University, for his assistance in the histological A thin chorion and scanty cortical alveoli are examination. among the peculiarities of oogenesis in viviparous rockfish. In the chorion of viviparous teleosts, a reduction in thickness is common and is correlated REFERENCES with the mode of reproduction.50 In the viviparous rockfish showing intraluminal gestation, the only 1. Kusakari M. Studies on the reproductive biology and artificial juvenile production of kurosoi Sebastes schlegeli. Sci. function of the chorion may be to separate each Rep. Hokkaido Fish. Exp. Stn 1995; 47: 41–124. embryo in the ovarian cavity during gestation. 2. Mizue K. Studies on a scorpaenous fish Sebastiscus marm- Because the cortical alveoli play some important oratus Cuvier et Valenciennes. V. On the maturation and the roles in the fertilization of eggs (e.g. formation of seasonal cycle of the ovaries of the marine ovoviviparous the perivitelline space, hardening of the fertiliza- teleost. Bull. Fac. Fish. Nagasaki Univ. 1959; 8: 84–110. tion membrane, sperm agglutination for prevent- 3. Moser HG. Seasonal histological changes in the gonads of ing polyspermy), they contain various substances Sebastodes paucispinis Ayres, an ovoviviparous teleost such as polysaccharides, sialoglycoproteins, and (Family Scorpaenidae). J. Morph. 1967; 123: 329–354. some enzymes.50 In the fertilized eggs of black 4. Takemura A, Takano K, Takahashi H. Reproductive cycle of rockfish the perivitelline space is very narrow and a viviparous fish, the white-edged rockfish, Sebastes taczanowskii. Bull. Fac. Fish. Hokkaido Univ. 1987; 38: 111–125. the fertilization membrane is flexible, changing its 5. Takemura A, Hara A, Takano K. Immunochemical identifi- shape from spherical to oval during late embryonic cationo and partial characterization of female-specific 1 development. It seems that such characteris- serum proteins in white-edged rockfish, Sebastes tacza- tics are due to a lack of cortical alveoli in this nowskii. Environ. Biol. Fish. 1991; 30: 23–29. species, and constitute a method of adaptation to 6. Takano K, Takemura A, Furihata M, Nakanishi T, Hara A. viviparity. Annual reproductive and spawning cycles of female The hormonal regulation of gametogenesis in Sebastes marmoratus. Environ. Biol. Fish. 1991; 30: 39–48. viviparous rockfish is almost similar to that in ovip- 7. Nagahama Y, Takemura A, Takano K, Adachi S, Kusakari M. arous species. Moreover, in viviparous rockfish Serum steroid hormone levels in relation to the reproduc- showing lecithotrophy, it is suggested that E2 tive cycle of Sebastes taczanowskii and S. Schlegeli. Environ. induces hepatic Vg synthesis, DHP induces final Biol. Fish. 1991; 30: 31–38. 8. Takemura A, Takano K, Yamauchi K. The in vitro effects of oocyte maturation in female fish, and 11-KT regu- various steroid hormones and gonadotropin on oocyte lates spermatogenesis in male fish. Remarkably maturation of the viviparous rockfish, Sebastes tacza- high female serum DHP levels during gestation nowskii. Bull. Fac. Fish. Hokkaido Univ. 1989; 40: 1–7. may suggest some important role in the mainte- 9. Nagahama Y, Adachi S. Identification of maturation-induc- nance of gestation. Further investigation is needed ing steroid in a teleost, the amago salmon (Oncorhynchus into the roles of DHP in pregnant female fish and rhodurus). Dev. Biol. 1985; 109: 428–435. spermatogenic male fish in viviparous teleosts. 10. Yamauchi K, Adachi S, Miura T. Endocrinological control of There is a discrepancy of approximately gonadal development. In: Tabeta O (ed). Early Life History 5 months between the reproductive cycles of and Prospects of Seed Production of Japanese Eel Anguilla female and male black rockfish. Such a phenome- japonica. Koseishya-Koseikaku, Tokyo. 1996; 65–84. 11. Billard R, Fostier A, Weil C, Breton B. Endocrine control of non is widely found in the genus Sebastes,25,51 and spermatogenesis in teleost fish. Can. J. Fish. Aquat. Sci. seems to be due to the male reproductive strategy 1982; 39: 65–79. in evolutional adaptability and to physiological 12. Miura T, Yamauchi K, Takahashi H, Nagahama Y. Hormonal changes in the response of male endocrine systems induction of all stages of spermatogenesis in vitro in the to their environments. The mechanisms underly- male Japanese eel (Anguilla japonica). Proc. Natl Acad. Sci. ing this discrepancy between the reproductive USA 1991; 88: 5774–5778. cycles of female and male black rockfish remain a 13. Ueda H, Young G, Crim LW, Kambegawa A, Nagahama Y. challenging area for further study. 17a,20b-dihydroxy-4-pregnen-3-one: plasma levels during sexual maturation and in vitro production by the testes of amago salmon (Oncorhynchus rhodurus) and rainbow trout (Salmo Gairdneri). Gen. Comp. Endocrinol. 1983; 51: 106– ACKNOWLEDGMENTS 112. 14. Miura T, Yamauchi K, Takahashi H, Nagahama Y. The role We wish to express our gratitude to Dr K Hirose, of hormones in the acquisition of sperm motility in salmo- Councilor of Japan Sea-Farming Association, for nid fish. J. Exp. Zool. 1992; 261: 359–363. critical reading of the manuscript. We are grateful 15. Ueda H, Kambegawa A, Nagahama Y. In vitro 11-ketotest- to the staff of the Japan Sea-Farming Association, osterone and 17a,20b-dihydroxy-4-pregnen-3-one produc- 922 FISHERIES SCIENCE H Mori et al.

tion by testicular fragments and isolated sperm of rainbow eroclitus IV. Yolk-vesicle formation. J. Exp. Zool. 1986; 239: trout, Salmo gairdneri. J. Exp. Zool. 1984; 231: 435–439. 277–288. 16. Miura T, Ninzeki M, Hirai H, Yamauchi K. Induction of final 34. Sorokin VP. The redfish; gametogenesis and migrations of maturation by the injection of chum salmon pituitary the Sebastes marinus (L.) and Sebastes mentella Travin. homogenate in the male Japanese huchen (Hucho perryi). Cons. Perm. Int. Explor. Mer. Rapp. Proc. Verb. 1961; 150: Bull. Fac. Fish. Hokkaido Univ. 1991; 42: 16–25. 245–250. 17. Mizue K. Studies on a scorpaenous fish Sebastiscus marm- 35. Hayashi S. Studies of the ultrastructural changes of ovarian oratus Cuvier et Valenciennes. II. The seasonal cycle of tissue with oogenesis and pregnancy in white-edged rock- mature testis and the spermatogenesis. Bull. Fac. Fish. fish, Sebastes taczanowskii. Master Thesis, Hokkaido Uni- Nagasaki Univ. 1958; 6: 27–38. versity, Hakodate, 1990. 18. Mizue K. About the seasonal cycle of mature testis of 36. Bromage NR, Whitehead C, Breton B. Relationships Sebastes inermis. Bull. Fac. Fish. Nagasaki Univ. 1959; 8: between serum levels of gonadotropin, oestradiol-17b, and 111–122. vitellogenin in the control of ovarian development in the 19. Igarashi T. Ecological studies on a marine ovoviviparous rainbow trout. II. The effects of alterations in environmen- teleost, Sebastes taczanowskii (Steindachner). I. Seasonal tal photoperiod. Gen. Comp. Endocrinol. 1982; 47: 366– changes of the testis. Bull. Fac. Fish. Hokkaido Univ. 1968; 376. 19: 19–26. 37. Koya Y, Soyano K, Yamamoto K, Obana H, Matsubara T. 20. Sasaki T, Igarashi T. Seasonal changes of the testis and the Oocyte development and serum profiles of vitellogenin and spermatogenesis of Sebastes vulpes Steindachner et Doder- steroid hormone levels in captive female Pacific herring lein. Bull. Fac. Fish. Hokkaido Univ. 1974; 25: 100–106. Clupea pallasii during their first maturational cycle. Fish. 21. Mancini G, Varbonara AO, Heremans JF. Immunochemical Sci. 2003; 69: 137–145. quantification of antigens by single radial immunodiffu- 38. Moore RK, Scott AP, Collines PM. Circulation C-21 steroids sion. Immunochemistry 1965; 2: 235–254. in relation to reproductive condition of a viviparous marine 22. Lowry OH, Rosebrough NJ, Farr AL, Randal RJ. Protein mea- teleost, Sebastes rastrelliger (grass rockfish). Gen. Comp. surement with the Folin phenol reagent. J. Biol. Chem. 1951; Endocrinol. 2000; 117: 268–280. 193: 265–272. 39. Koya Y, Hamatsu Y, Matsubara T. Testicular development of 23. Asahina K, Kambegawa A, Higashi T. Development of male Sebastolobus macrochir in the Pacific coast of south- microtiter plate enzyme-linked immunosorbent assay for eastern Hokkaido. Bull. Hokkaido Natl Fish. Res. Inst. 1994; 17a,20b,21-trihydroxy-4-pregnen-3-one, a teleost gonadal 58: 1–8. steroid. Fish. Sci. 1995; 61: 491–494. 40. Muñoz M, Casadevall M, Bonet S. Annual reproductive 24. Korsgaard B, Petersen I. Vitellogenin, lipid and carbohy- cycle of Helicolenus dactylopterus dactylopterus (Teleo- drate metabolism during vitellogenesis and pregnancy, and stei: Scorpaeniformes) with special reference to the ova- after hormonal induction in the blenny Zoarces viviparus ries sperm storage. J. Mar. Biol. Assoc. UK 1999; 79: 521– (L.). Comp. Biochem. Physiol. 1979; 63B: 245–251. 529. 25. Takahashi H, Takano K, Takemura A. Reproductive cycles of 41. Hunt SMV, Simpson TH, Wright RS. Seasonal changes in Sebastes taczanowskii, compared with those of other rock- the levels of 11-oxotestosterone and testosterone in the fishes of the genus Sebastes. Environ. Biol. Fish. 1991; 30: serum of male salmon. Salmo salar L. and their relation- 23–29. ship to growth and maturation cycle. J. Fish Biol. 1982; 20: 26. Koya Y, Inoue M, Naruse T, Sawaguchi S. Dynamics of 105–119. oocyte and embryonic development during ovarian cycle of 42. Kime DA, Manning NJ. Seasonal patterns of free and con- the viviparous mosquitofish Gambusia affinis. Fish. Sci. jugated androgens in the brown trout Salmo trutta. Gen. 2000; 66: 63–70. Comp. Endocrinol. 1982; 48: 222–231. 27. Wourms JP. Viviparity: the maternal-fetal relationship in 43. Kobayashi M, Aida K, Hanyu I. Annual changes in plasma fishes. Am. Zool. 1981; 21: 473–515. levels of gonadotropin and steroid hormones in goldfish. 28. Boehlert GW, Yoklavich MM. Reproduction, embryonic Nippon Suisan Gakkaishi 1986; 52: 1153–1158. energetics, and the maternal–fetal relationship in the vivip- 44. Cochran RC. Serum androgens during the annual repro- arous genus Sebastes. Biol. Bull. 1984; 167: 354–370. ductive cycle of the male mummichog, Fundulus heterocli- 29. Boehlert GW, Kusakari M, Shimizu M, Yamada J. Energetics tus. Gen. Comp. Endocrinol. 1987; 65: 141–148. during embryonic development in kurosoi, Sebastes 45. Koya Y, Soyano K, Yamamoto H, Obana H, Matsubara T. schlegeli Hilgendorf. J. Exp. Mar. Biol. Ecol. 1986; 101: 239– Testicular development and serum profiles of steroid hor- 256. mone levels in captive male Pacific herring Clupea pallasii 30. Takemura A, Takano K, Takahashi H. The uptake of macro- during their first maturational cycle. Fish. Sci. 2002; 68: molecular materials in the hindgut of viviparous rockfish 1099–1105. embryos. J. Fish. Biol. 1995; 46: 485–493. 46. Ueda H, Hiroi O, Hara A, Yamauchi K, Nagahama Y. 31. Takano K. Ovarian structure and gametogenesis. In: Changes in serum concentrations of steroid hormones, thy- Takashima F, Hanyu I (eds). Reproductive Biology of Fish roxin, and vitellogenin during spawning migration of the and Shellfish. Midori-Shobo, Tokyo. 1989, 3–34. chum salmon, Oncorhynchus keta. Gen. Comp. Endocrinol. 32. Yamamoto K. Studies on the formation of fish eggs. VII. The 1984; 53: 203–211. fate of the yolk vesicle in the oocytes of the herring, Clupea 47. Truscott B, Idler DR, So YP, Walsh M. Maturational steroids pallasii, during vitellogenesis. Annotat. Zool. Japon. 1956; and gonadotropin in upstream migratory sockeye salmon. 29: 91–97. Gen. Comp. Endocrinol. 1986; 62: 99–110. 33. Selman K, Wallace RA, Barr V. Oogenesis in Fundulus het- 48. Kobayashi M, Aida K, Hanyu I. Gonadotropin surge during Reproductive cycle of black rockfish FISHERIES SCIENCE 923

spawning in male goldfish. Gen. Comp. Endocrinol. 1986; 50. Iwamatsu T. Fertilization in fishes. In: Tarín JJ, Cano A (eds). 62: 70–79. Fertilization in Protozoa and Metazoan Animals. Springer- 49. Miura T, Yamauchi K, Takahashi H, Nagahama Y. Involve- Verlag, Berlin. 2000; 89–145. ment of steroid hormones in gonadotropin-induced testic- 51. Echeverria TW. Thirty-four species of California rockfishes: ular maturation in male Japanese eel (Anguilla japonica). maturity and seasonality of reproduction. Fish. Bull. 1987; Biomed. Res. 1991; 12: 241–248. 85: 229–250.