Caspase-3在黄鳝性腺性逆转过程中的表达及定位分析

为探究黄鳝( Monopterus albus )Caspase-3在性腺性逆转发育过程中的功能和作用,实验扩增黄鳝 caspase-3 的部分序列,检测了其在不同发育时期性腺中mRNA表达水平、蛋白相对含量以及表达位置。通过PCR扩增获得黄鳝 caspase-3 基因cDNA序列,推导的氨基酸序列对比发现,黄鳝 caspase-3 与大黄鱼( Larimichthys crocea )和鳜( Siniperca chuatsi )亲缘关系最近。实时定量PCR ( qRT-PCR)结果显示, caspase-3 mRNA在黄鳝各发育时期性腺中均有表达,在卵黄生成期性腺中表达量最高,并伴随性腺向雄性发育表达量呈下降的趋势;而其蛋白相对含量在性腺性逆转过程中呈现逐渐上升的趋势,间性晚期最高。免疫组织化学染色结果显示,Caspase-3蛋白阳性信号定位于卵黄生成期卵母细胞细胞质、皮质小泡期卵母细胞的细胞核和颗粒细胞,以及各个发育时期性腺中初级生长期卵母细胞的细胞质和细胞核。综上,Caspase-3与黄鳝性逆转过程关系密切,推测其可能参与了性逆转过程中卵母细胞的凋亡过程。     

Effects of aromatizable and nonaromatizable androgens on the sex inversion of red-spotted grouper (Epinephelus akaara)

The effects of aromatizable 17α-methyltestosterone (MT) and non-aromatizable 17α-methyldihydrotestosterone (MDHT) on sex inversion in red-spotted grouper, Epinephelus akaara, were investigated. Fish were implanted with MT, MDHT and MT+AI (aromatase inhibitor, AI) respectively for one month. The results showed that the three treated groups turned into transitional stage with intersex gonads, which contained atretic oocytes and spermatogenic germ cells at all stages of spermatogenesis. The controls did not change sex. The gonads of more than half MT-implanted fish were in early transitional stages of sex inversion, whereas those of more than half MDHT and MT+AI-implanted fish were in late transitional stages of sex inversion. No difference in serum estradiol-17β (E₂) levels between the controls and the treated groups were observed, whereas 11-ketotestosterone (11-KT) and testosterone (T) levels increased in all treated groups. Significantly lower gonadosomatic index (GSI) and gonadal aromatase activity were observed in the treated groups, which were in accordance with the lower mRNA expression of P450aromA. However, P450aromB mRNA expression increased in the MT group, while it did not change in the MDHT group. These results suggest that the sex inversion of red-spotted grouper by MT and MDHT implantation might be due to the suppression of P450aromA gene expression, and resulting in both the decrease of the ovarian estrogen –secretion, as well as the increase in the 11-KT levels. Furthermore, the main reason for MT being less effective than MDHT might be due to partial aromatization of MT to estrogen.     

半滑舌鳎性腺的组织学研究

采用组织切片,对半滑舌鳎性腺的组织学结构进行了观察研究。结果表明,半滑舌鳎的精巢为小叶型,精巢内的生殖细胞分为五种。其精原细胞存在于精小叶的内壁上,精母细胞和成熟的精子细胞位于精小囊中。精子成熟后,精小囊破裂释放精子进入小叶腔,完成发育过程。半滑舌鳎卵巢外有被膜包裹,其被膜的结缔组织可明显地分为内外两层。内层的结缔组织、血管与原始生殖细胞一同伸进卵巢内部形成许多产卵板。产卵板以卵巢腔为中心向四周呈辐射状排列,上有原始生殖细胞分化形成卵原细胞和不同时相的卵母细胞。半滑舌鳎卵巢中的卵母细胞的发育可以分为5个时相。本文描述了各时相卵母细胞的形态学特征。并阐述了半滑舌鳎的产卵类型。     

Diurnal rhythm of serum steroid hormone levels in the Japanese whiting,Sillago japonica,a daily-spawning teleost

Ovarian developmental stages and serum steroid hormone levels were examined at six different times of day (0100, 0600, 1000, 1300, 1600, 2000 h) in a marine teleost, the Japanese whiting Sillago japonica, which has an asynchronous-type ovary containing oocytes at various stages of development and spawns every day during a period ranging up to three months. The largest oocytes in the ovaries at the active vitellogenic or post-vitellogenic stages were found between 0100 and 1300 h. Oocyte maturation indicated by germinal vesicle breakdown (GVBD) occurred at 1600 h, and ovulated oocytes were observed in the ovaries collected at 2000 h. These processes were accompanied by a significant daily change in serum steroid hormone levels. The serum level of estradiol-17β showed a peak in fish with mature oocytes sampled at 1600 h. In these fish, the second-largest oocytes in the ovaries were at the initial stage of vigorous vitellogenesis, the secondary yolk stage. Therefore the highest level of serum estradiol-17β was considered to be due to the second-largest oocytes. Testosterone levels remained low and constant throughout the experimental period. The serum levels of 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-diOHprog) peaked at 1600 h at which time all fish had mature oocytes. These results indicate that the Japanese whiting possesses a diurnal rhythm of oocyte development including vitellogenesis, oocyte maturation and ovulation, and further suggest that daily cycles in oocyte growth and maturation which simultaneously take place in an ovary are regulated by diurnal secretions of estradiol-17β and the maturation-inducing steroid, 17α,20β-diOHprog.     

Treatment with an aromatase inhibitor induces complete sex change in the protogynous honeycomb grouper (Epinephelus merra)

To study the mechanism of protogynous sex change in honeycomb grouper, the wild fish of both sexes and transitionals were collected for one year, and changes in gonadal structures and serum levels of sex hormones in each individual were examined. The onset of sex change was associated with low serum estradiol - 17β (E2) levels. In order to clarify whether E2 deprivation causes sex change, maturing females were implanted with the non-steroidal aromatase inhibitor (Fadrozole, AI). AI caused complete sex change from female to functional males in two and half months. The present results suggest that E2 is an important endogenous factor which regulates protogynous sex change in the honeycomb grouper.     

Brain steroid contents in the catfish Heteropneustes fossilis: sex and gonad stage-specific changes

Neurosteroids are those which are synthesized in the central nervous system independently of supply by peripheral endocrine glands. In the present study, brain contents of the steroid hormones, estradiol-17β (E(2)), testosterone (T), corticosteroids, and progestins were investigated in both male and female catfish Heteropneustes fossilis in prespawning (vitellogenic) and spawning (post-vitellogenic) phases using ELISA or HPLC. The data show that the measured steroid hormones showed both stage-specific and sex-related variations. Brain E(2) was significantly higher in males in the prespawning phase and in females in the spawning phase. Testosterone was significantly higher in males in comparison with females in the prespawning phase. Cortisol was significantly higher in the prespawning and spawning phases in males than in females. Corticosterone level was low in the brain. 21-deoxycortisol and deoxycorticosterone were significantly higher in the prespawning phase than in the spawning phase. Male brain recorded the highest concentration of deoxycorticosterone. Progesterone (P(4)) was high in the prespawning phase and low in the spawning phase in both sexes. Levels of 17-hydroxy-4-pregnene-3,20-dione and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-DP) and the metabolites of P(4) were the highest in females in the prespawning phase. The stage-specific and sexual differences in the content of the steroids suggest their biosynthesis in the brain, which may have implications in brain functions, in addition to reproductive regulation.     

Annual changes in ovarian development and plasma estradiol-17β level in reared female common Japanese conger, <Emphasis Type="Italic">Conger myriaster</Emphasis>

ABSTRACT:   Annual changes in the gonadosomatic index (GSI), oocyte diameter, gonadal histology and plasma estradiol-17β (E₂) levels were investigated in female common Japanese conger Conger myriaster in captivity. Juveniles were caught in September 1999 and reared in seawater at temperatures ranging from 10–20°C for 3 years. All fish were immature when captured in September 1999. GSI and oocyte diameter increased from October 2000, peaked between summer and autumn 2001, and bottomed-out in winter 2001. Plasma E₂ level also increased from October 2000, but remained high. The ovarian developmental stage was at the peri-nucleolus stage or the oil droplet stage until September 2000. Vitellogenesis started in October 2000 and oocytes progressed to the tertiary yolk globule stage by summer 2001. However, vitellogenic oocytes regressed in all females after autumn 2001. The neogenetic oocytes began to increase after November 2001 and ovarian development progressed in 2002 as they did in 2001, although maximum GSI in 2002 was half its 2001 value. These data indicate that ovarian development in the common Japanese conger has an annual periodicity, and that these congers may be able to spawn in multiple years under rearing condition.     

The timing of puberty in cultured female yellowtail flounder, Limanda ferruginea (Storer): Oogenesis and sex steroid production in vivo and in vitro

This study examines puberty in female yellowtail flounder, correlating macroscopic, histological and endocrine parameters at the gonadal level. Short-term ovarian tissue incubations were used to study estradiol-17β production in relation to oogenic stage. Examining 2 year classes of young flounder demonstrated that cultured females retain phenotypic plasticity in reproductive age with pubertal onset occurring in one-year, two-year and three-year-olds. Immature ovaries were steroidogenic and capable of responding to gonadotropic stimulation. Endocrine puberty in females was detected by a peak in estradiol-17β production during the cortical alveolar oocyte stage prior to any ovarian evidence of vitellogenesis. Puberty, once initiated, proceeded to ovulation within 8 to 12 months; vitellogenic oocyte development followed the group synchronous pattern. Estradiol-17β was clearly the dominant of the two measured plasma hormones during pubertal onset and throughout vitellogenesis. Plasma testosterone was consistently detectable at low levels by mid-to late-vitellogenesis. Ovaries showed the highest sensitivity to gonadotropic stimulation in vitro during late-vitellogenesis. Variable plasma levels in both estradiol-17β and testosterone occurred in preovulatory and ovulating females during the captive spawning period. Together the results show that yellowtail flounder can mature at a young age and small size when culture conditions permit. In addition, the early sensitivity to gonadotropin by the immature ovaries may be an important physiological determinant for the timing of puberty in this species.     

Sex differences in circulating steroid hormone levels in the red drum, Sciaenops ocellatus L.

Steroid profiles of cultured and captive red drum (Sciaenops ocellatus L.) were investigated to evaluate the potential use of circulating sex steroid levels as a tool for gender identification in this species. Cultured 18‐month‐old fish were maintained on a 120‐day shortened photothermal cycle to induce precocious maturation. Additionally, wild‐caught fish were maintained in captivity under simulated natural photothermal conditions from late spring to early fall. Circulating 11‐ketotestosterone (11‐KT) levels were significantly higher in males compared with females during the early stages of gonadal growth in both cultured and captive fish. Plasma testosterone (T) levels showed a similar trend; however, the differences were significant only when males were already producing sperm. 17β‐estradiol (E₂) concentrations were low in males and females before gonadal recrudescence but increased significantly with the progression of vitellogenesis in females. These results show that a test using a minimum concentration of circulating 11‐KT could be developed to differentiate between sexes in the early stages of gonadal maturation in red drum. Moreover, plasma E₂ concentrations could be used to identify vitellogenic females. The two steroids considered together could help avoid possible error in gender identification due to unusually high levels of certain steroids encountered in some individuals.     

Gonadal Stage and Sex Steroid Correlations in Siberian Sturgeon,Acipenser baeri,Habituated to a Semitropical Environment

Stages of gonadal development, in association with plasma concentrations of the sex steroids 17β‐estradiol (E2), progesterone (P4), testosterone (T), and 11‐ketotestosterone (11‐KT), were investigated for a single time point during a natural breeding season in 7‐yr‐old Siberian sturgeon, Acipenser baeri Brandt, exposed lifelong to a warmwater environment. Among females, examination of gonadal tissue showed variation in ovarian stage, with 12.5, 47.5, 22.5, and 17.5% of females found at Stages 2 (previtellogenic), 3 (early vitellogenic), 4 (mid‐vitellogenic), and 5 (migratory nucleus), respectively. Although patterns varied among the hormones, plasma concentrations of E2, T, and 11‐KT became increasingly elevated in females as maturation progressed. On the basis of histological criteria, males were classified as either premeiotic (quiescent) or meiotic and 50% of the males sampled were found at each stage. Significant elevations in circulating concentrations of plasma E2 and T were observed in meiotic versus premeiotic males, and there was a rise in plasma 11‐KT concentration that approached significance (P = 0.056).     

A histological study of gametogenesis in captive red snapper Lutjanus campechanus

Gametogenesis was monitored histologically in wild‐caught red snapper (Lutjanus campechanus, Poey) maintained in captivity under simulated natural photothermal conditions. Gonads were collected every 2–3 weeks (average n = 14) for histology during the pre‐spawning season (February to May, temperature increasing from 16°C to 24°C). Primary vitellogenic oocytes were first observed in one female when temperature reached 20°C. Subsequent samples revealed females in pre‐vitellogenesis or at early stages of vitellogenesis, although one female had tertiary vitellogenic (Vtg3) oocytes. The first histological signs of spermatogenesis were observed when temperature reached 17°C. Spermatozoa were observed in testicular lobules of all males sampled on 14 May (24°C) but little or no sperm was released during manual stripping. Ten males and 10 females were left in tanks and monitored for spawning. No egg release was observed during the monitoring period that encompassed the natural spawning season of wild red snapper. Ovarian biopsies taken during the late spawning season (16 July) revealed that four of eight sampled females had Vtg3 oocytes. Males were manually stripped but released no sperm. These results indicate that captive red snapper can complete gametogenesis in photothermal controlled systems. Additional research is needed to develop procedures that will achieve reliable maturation and spawning.     

Preliminary Observations on the Reproductive Physiology of Female Orangemouth Corvina in Captivity

Seasonal changes In oocyte growth and plasma estradiol, testosterone, thyroid hormones and vitellogenin levels were monitored in three captive adult female orangemouth corvina Cynoscion xanthulus subjected to a condensed (8 mo) seasonal cycle of photoperiod and water temperature. Plasma concentrations of estradiol and testosterone began to rise when temperature increased to 28 C and photoperiod to 15 h light (midsummer conditions). This was accompanied by elevated circulating levels of vitellogenin and the appearance of vitellogenic oocytes (diameter > 100 μm). Estradiol concentrations and mean oocyte size increased concurrently during late summer conditions and were maximum during fall conditions, approximately 8 wk after the beginning of ovarian recrudescence. In contrast, plasma levels of thyroid hormones did not show any distinct seasonal changes. Gonadally recrudesced females contained several size classes of vitellogenic oocytes. Injection of these fish with a luteinizing hormone-releasing hormone analog (LHRHa) caused maturation and spawning of the largest oocytes (mean diameter of follicles × 2500 μm). Another group of vitellogenic oocytes had been recruited into this size class by 2 wk after the end of spawning, which suggests that this species is capable of repeated spawning during the reproductive season. Injection of LHRHa resulted in increased plasma levels of gonadotropin and repeated spawning over several days. LHRHa-induced final oocyte maturation, ovulation and spawning were preceded by increases in plasma levels of two teleostean maturation-inducing steroids, 17α,20β, 21-trihydroxy-4-pregnen-3-one (20β-S) and 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-P₄). The results provide preliminary evidence that oocyte growth is stimulated in orangemouth corvina subjected to an abbreviated seasonal cycle in captivity under midsummer photoperiod and temperature conditions and is associated with seasonal increases in plasma estradiol concentrations and vitellogenin production. However, the relative importance of 20β-S versus 17α, 20β-P₄ in the control of final oocyte maturation in this species could not be determined from the results of the present study.     

Annual sex hormonal profiles, gonad development and age determination of the Mekong giant catfish (Pangasianodon gigas, Chevey)

Annual sex hormonal profiles, gonad development and age determination of 18 (13–14 kg body weight) and three (145–226 kg body weight) Mekong giant catfish (MGC) (Pangasianodon gigas, Chevey) reared in earthen ponds in Chiang Mai and from the Mekong River in Chiang Rai, Thailand, respectively, were investigated. The hormonal profiles were determined from blood samples of the fish by electrochemiluminescence immunoassay during May to August in 2000. The highest testosterone levels of 0.06 ng mL^?1 in both sexes and the highest oestradiol of 47.8 and 14.23 pg mL^?1 in females and males, respectively, were observed in May. The gonadosomatic index was found to be 0.07 for males and 0.38 for females from ponds in comparison with 2.27 for males and 8.29 for females from the Mekong River. Higher development stages of spermatocytes and oocytes of the cultured fish in May than in February and November were demonstrated. No mature germ cells were obtained from either the males or females, indicating no sexual maturity. The average age of fish was determined from the annual rings of the cross‐section of the pectoral fins by a stereomicroscope. The average age of fish in earthen pond determined from the annual ring was 8 years, which agreed with the actual culture records, while those from the Mekong River were 21 years. This information will be beneficial for the breeding programmes and conservation of the MGC.     

粗唇鮠精巢显微结构和精子发生的研究

采用光镜和透射电镜观察了粗唇鮠(Leiocassis crassilabris)精巢结构和精子发生。光镜结果显示:粗唇鮠的精巢各小叶由数个小囊组成。从第Ⅱ期到第V期时,初级精母细胞逐渐发育成为次级精母细胞、精子细胞和精子,第Ⅵ期为排精后退化时期。透射电镜观察粗唇鮠的精原细胞、初级精母细胞、次级精母细胞、精子细胞和精子的形态以及细胞核和线粒体等的结果表明:在精子发生各期中线粒体和细胞核有明显的变化;精原细胞时期细胞器较丰富,到精子细胞后期,细胞器的形态和数量发生了较大变化;随着精母细胞的发育,核质凝聚程度逐渐增强;粗唇鮠的精子具有椭圆的头部和复杂的中片,并具有由外膜折叠形成的波浪形的结构。     

Seasonal changes in plasma gonadal steroid concentrations and gonadal morphology of male and female tench (Tinca tinca, L.)

In order to gain a better understanding of the reproductive cycles of male and female tench (Tinca tinca), gonadosomatic index, gonad histology and plasma concentrations of estradiol‐17β (E₂), testosterone, an drostenedione, 11‐ketotestosterone (11‐KT), 17,20β, 21‐trihydroxy‐4‐pregnen‐3‐one (17,20β,21‐P), 17,20β‐dihydroxy‐4‐pregnen‐3‐one (17,20β‐P) and 17,20α‐dihydroxy‐4‐pregnen‐3‐one (17,20α‐P) were measured at the four seasons of the year, plus a further sampling coincident with the peak of spawning in early July. As expected, in both males and females, the plasma concentrations of androgens (excluding 11‐KT in females – undetectable) and C₂₁ steroids were significantly more elevated in the spring and summer (when most gonadal development took place) than in the autumn and winter. The only unexpected finding was that 17,20β‐P and 17,20β,21‐P, the steroids that are normally associated with oocyte final maturation in females and spermiation in males, were found in substantial amounts in both pre‐vitellogenic, pre‐spermatogenic and post‐spawning fish. This suggests that these steroids may have other as yet unidentified roles in this species.     

Aromatase (P450arom) and 11β-hydroxylase (P45011β) genes are differentially expressed during the sex change process of the protogynous rice field eel, monopterus albus

Steroids are known to play a crucial role in gonadal sex differentiation in many non-mammalian vertebrates, but also in the gonadal sex change of hermaphroditic teleosts. We investigated the expression of two genes encoding key steroidogenic enzymes, i.e., cytochrome P450 aromatase (P450arom) and cytochrome P45011β-hydroxylase (P45011β), during the sex change of the protogynous rice field eel, Monopterus albus. Using RT-PCR with degenerate primers, we cloned rice field eel homologous fragments for both genes (rcP450arom and rcP45011β) as indicated by the high level of homology with P450arom and P45011β sequences from various vertebrates. Gonadal expression of rcP450arom and rcP45011β mRNA levels were then assessed during the sex change by semi-quantitative RT-PCR and a real-time RT-PCR. rcP450arom was predominantly expressed in ovary, much less in ovotestis, and barely in testis. Conversely, P45011β was markedly up-regulated at the onset of testicular development. These findings underline that regulation of steroidogenesis is an important process in the sex change of protogynous rice field eel, and they clearly indicate that the concomitant down-regulation of P450arom and up-regulation of P45011β are of pivotal importance to the sex change of this species.     

Artificial sex reversal of white grouper (Epinephelus aeneus) utilizing aromatase inhibitor (Fadrozole)

The white grouper is a desirable aquaculture species that adapts to captivity, grows well and commands a high market price. However, little is known about reproductive biology or control of sex reversal of this protogynous hermaphrodite. In this study, female white groupers were implanted with one dose of 17α‐methyltestosterone (10 mg/kg body weight [BW], MT) and two doses of aromatase inhibitor, fadrozole (1 and 3 mg/kg BW, FD1 and FD3) once a month for 4 months (April–July). At the start of the study, the fish had gonads full of oocytes compared to the end of the experiment when the control group mature oocytes compared to the experimental groups MT, FD1 and FD3 that exhibited different stages of testicular tissue. Plasma levels of testosterone were significantly highest in the FD3 group and the highest 11‐Ketotestosterone levels were observed in the MT group. Plasma levels of oestradiol (E₂) were significantly lower in the FD implanted groups, compared to initial individuals and control groups. The use of aromatase inhibitor, FD for sex reversal both gives further insight into the mechanisms controlling sex differentiation and provides an alternative to steroid treatment.     

Intra‐annual changes in reproductive indices of male and female Himalayan snow trout,Schizothorax richardsonii (Gray, 1832)

Periodic changes in reproductive hormone levels, gonadal histology and gonadosomatic index (GSI) of snow trout, Schizothorax richardsonii, were examined to ascertain annual cycle of gonadal development and reproductive status in their natural habitat. In females, there were coherent changes in plasma 17β‐oestradiol and vitellogenin along with GSI, oocyte maturation and vitellogenic progression, collectively indicating two distinct maturation peaks during the months of September and February. Coinciding with this, in males, plasma 11‐keto testosterone was also noticeably higher during September and February, with highest GSI values in September. However, plasma 17α, 20β‐dihydroxyprogesterone levels in males were found to be persistently high from September to February. This observation suggests the potential presence of matured oozing males over a longer period, unlike in females. Overall, the close association between reproductive hormone levels, GSI and gonadal maturation stages in males and females (particularly, the presence of postovulatory follicle complexes) with apparent natural synchronization clearly indicates that S. richardsonii breeds twice in a year, possibly during late September to early November and late February to early April in the coldwater riverine habitats of the Indian Himalayan region.