Role of the sertoli cell in spermatogenesis: TheSqualus testis model

In the dogfish sharkSqualus acanthias different germ cell stages are topographically segregated within the testis. Using this species we have developed methods for the isolation and culture of Sertoli cells from premeiotic, meiotic and post-meiotic stages of spermatogenesis and present preliminary evidence for stage-dependent variations in cell morphology and behavior, thymidine incorporation, protein synthesis and steroidogenesis. The goal of future studies is to determine how maturational changes are regulated in Sertoli cells and, in turn, to elucidate Sertoli cell-germ cell interactions.     

On the blood volume of the Mediterranean dogfish,Scyliorhinus canicula

The blood volume of the Mediterranean dogfish,Scyliorhinus canicula was determined to be 4.06±0.92 ml/100 g body weight. This result is discussed in relation with the dye dilution technique used, the degree of dye binding with circulating plasma proteins and the values and techniques used by other authors.     

Stimulatory and inhibitory regulation of DNA synthesis during spermatogenesis: studies in Squalus acanthias

Spermatogenesis is a unique developmental sequence involving multiple cell to cell interactions and several categories of regulatory molecules. In contrast to conventional mammalian models in which testicular organization is highly complex, the testis of the dogfish shark Squalus acanthias is technically advantageous for elucidating stage-dependent structural and functional charactericsics and for in vitro regulatory studies. Using incorporation of [³H]thymidine into acid-insoluble molecules as a measure of DNA synthesis by spermatocysts (germ cell/Sertoli cell units) of premeiotic stages, we obtained evidence of a growth inhibitory bioactivity (chalone) within the testis. This activity is differentially distributed (postmeiotic > meiotic > premeiotic), suggesting that more advanced developmental stages, which are upstream in the vascular pathway within the testis, may control the size of the proliferating spermatocyst population and, hence, the advance of less mature stages. These data provide direct evidence for humoral communication between stages of spermatogenesis.     

Reducing elasmobranch bycatch: Laboratory investigation of rare earth metal and magnetic deterrents with spiny dogfish and Pacific halibut

Spiny dogfish (Squalus acanthius) comprises a significant unwanted bycatch on demersal longlines set for halibut and cod in shelf waters of the east and west coasts of North America. In this laboratory study, attacks on baits were tested in the presence of two different rare earth materials (neodymium–iron–boride magnets and cerium mischmetal) believed to deter elasmobranch catch. Experiments were made with spiny dogfish and with Pacific halibut (Hippoglossus stenolepis) in pairwise tests of the rare earth materials and inert metal controls. Dogfish attacked and consumed baits tested with cerium mischmetal at a lower frequency than controls. Times to attack the baits were significantly higher in the presence of mischmetal, as were numbers of approaches before first attack. The time differential between mischmetal and control treatments and the number of baits consumed converged with increasing food deprivation (1 h, 2 d, and 4 d), but treatment differences were always significant. Cerium mischmetal appeared to be irritating to dogfish and may disrupt their bait detection and orientation abilities. Magnets also appeared to irritate dogfish but provided no protection for baits in feeding trials. Pacific halibut showed no reaction whatsoever to the rare earth magnets or cerium mischmetal. Mischmetal, therefore, may be useful in reducing spiny dogfish bycatch in the halibut fishery. Disadvantages in using mischmetal in commercial operations are expense, hazardous nature, and relatively rapid hydrolysis in seawater.     

Stimulation of gonadal and germ cell development in larval and juvenile carp (Cyprinus carpio L.) by homologous pituitary extract

The data presented show that in larval carp, gonadal size has increased distinctly after treatment with homologous pituitary extract (PE). Moreover, the precocious onset of primordial germ cell proliferation and of sex differentiation into male and female gonads was induced. Body weight of treated and control specimens did not differ significantly from each other throughout the experiment. Treatment of juvenile carp with homologous PE led also to an increase in gonadal size without concomitant increase in body weight. Induction of precocious spermatogenesis was observed too. GTH-levels in control and PE-treated carp were monitored by means of a homologous radioimmunoassay, showing that in PE-treated carp the GTH-level was distinctly elevated. The possible role of pituitary hormones in larval and juvenile gonadal development is discussed.     

外源性促性腺激素诱导日本鳗鲡精子发生和成熟的作用机制

用兔抗血清对抗促黄体素生成素受体（LHR）或称绒毛膜促性腺激素受体（CGR）和雄激素受体（AR）进行LHR和AR免疫组织化学定位,以揭示外源性促性腺激素（鲤脑垂体激素和hCG）诱发日本鳗鲡精子发生及其内分泌机制。结果表明,经过注射激素处理后的实验组与注射前的对照组相比较,其精巢发育和精子发生出现十分显著的变化。组织学切片观察显示,激素处理前鳗鲡精巢处于精原细胞增殖期,而两种激素混合注射后第10天,实验组可见精小叶中精原细胞的有丝分裂和初级与次级精母细胞的数量显著的增加。注射后第35天,靠近生殖上皮除有少量精原细胞外,精小叶中有大量初级精母细胞和次级精母细胞和少数精子细胞以及管腔中存在少量精子。在注射后第83天,日本鳗鲡完成了精子发生和精巢发育成熟以及释精。免疫组织化学染色结果进一步揭示,激素处理前,LH受体免疫活性分布在生殖上皮,显示强的免疫阳性反应;激素处理后,LH受体定位在Sertoli细胞和间质细胞以及精原细胞和初级与次级精母细胞的胞膜上,均显示强的免疫阳性反应。激素处理前,雄激素受体定位在生殖上皮和早期生精细胞的胞膜上;激素处理后,AR则定位在这些生精细胞的核或胞质,而精子细胞和精子显示免疫阴性反应。这些结果首次证明了这两种激素诱导鳗鲡精子发生和成熟的作用机制是通过LH受体和雄激素受体的介导。     

Ontogenetic changes of alpha-melanocyte-stimulating hormone (α-MSH)-like immunoreactivity in the brain and hypophysis of a scyliorhinid dogfish (Scyliorhinus torazame): An immunohistochemical study

Ontogenetic development of α-MSH-IR (alpha-melanocyte-stimulating hormone-like immunoreactivity) in the brain and hypophysis of a scyliorhinid dogfish (Scyliorhinus torazame) was studied immunohistochemically. α-MSH-IR first appeared in the hypothalamus of the brain in embryos at the 43-mm stage. Thereafter, α-MSH-positive cells and fibers gradually increased in number in mainly the tuberculum posterius of the hypothalamus during the embyonic periods. In the post-hatching juveniles at the 95-mm and 125-mm stages, α-MSH-positive structures in the brain were comparable to those in adult fish, although the distributional range of α-MSH-IR structures in the brain was less extensive than those in adult fish. In the hypophysis, α-MSH-positive cells first appeared in the caudal part of the adenohypophysis at the 54-mm stage, approximately coincident with the onset of pigmentation in the skin. During ontogenesis, the MSH-producing cells increased in number straightforwardly in the adenohypophysis. This revised version was published online in August 2006 with corrections to the Cover Date.     

Existence of APGWamide in the testis and its induction of spermiation in Haliotis asinina Linnaeus

Immunolocalization by using rabbit polyclonal antibody against APGWamide demonstrated that there was APGWamide immunoreactivity (APGW-ir) in the varicosed nerve fibers in the capsule, connective tissue and trabeculae of the testis whereas there was no APGW-ir in the ovary of sexually mature broodstocks of Haliotis asinina. In vivo bioassay, by injecting APGWamide into foot muscle of mature males and females (age over 24 months), showed that most males (87.50%) injected with 200 μl of 10^− 3 M APGWamide (170 μg APGWamide/animal) could be induced to spermiate whereas fewer males (43.75%) injected with 200 μl of 10^− 6 M APGWamide (0.17 μg APGWamide/animal) could spermiate. Following injection with 10^− 3 M, 53% of males spawned within 3 to 4 h, while the remaining males spawned within 2 to 3 h. The former tended to release more sperm at the range of 1.04 to 2 × 10⁹, and the latter at the range 3.62 × 10⁷ to 2.07 × 10⁸ cells, respectively, and that the sperm of the former group appeared to be more motile. Following the injection of 10^− 6 M, all inducible males spawned within 2–3 h, and 43% of spawned males have sperm around 2 × 10⁹, while 57% have fewer sperm number ranging from 2.75 × 10⁷ to 9.94 × 10⁸ cells. In contrast to males, none of the mature females with ripen ovary could be induced to ovulate. These data suggest that APGWamide is an important signaling molecule that plays a role in the male reproductive process that could be used to induce spermiation.     

Bilirubin-binding protein in the serum of spawning-migrating chum salmon,Oncorhynchus keta: Its identity with carotenoid-carrying lipoprotein

The serum carotenoid level gradually increased during the spawning migration of chum salmon (Oncorhynchus keta). Zeaxanthin, a yellowish carotenoid, was detected in the serum of spent males, while astaxanthin was a main carotenoid in the serum of males and females at other developmental stages. A high bilirubin level was found in the serum of spent males; the serum of spent male was yellow in colour due to the presence of zeaxanthin and bilirubin, although the serum of the other fish was bright orange.Three types of carotenoid-carrying lipoprotein such as low density lipoprotein, high density lipoprotein (HDL) and very high density lipoprotein fractions were present in the serum. Carotenoid-carrying lipoprotein from the HDL fraction became a main component during spawning migration. The serum bilirubin from the spent male salmon was distributed in the HDL fraction. Because bilirubin, as well as carotenoids, were present in the HDL fraction, the carotenoid-carrying lipoprotein from the HDL fraction was identical with bilirubin-binding protein in the serum of spent males. This carrier lipoprotein had two subunits (molecular weight 24,000 and 12,000); no disulfide bond was detected between the two subunits.     

谷氨酰胺对斜带石斑鱼GF-1细胞中C-Myc蛋白的表达与神经坏死病毒复制的影响

为探讨C-Myc表达、谷氨酰胺代谢和神经坏死病毒复制三者之间的关系,本研究首先克隆了斜带石斑鱼鳍条细胞(GF-1)中的C-Myc基因(GF-1-C-Myc),结果显示GF-1-CMyc基因cDNA全长814 bp,开放阅读框(ORF)为285 bp,编码95个氨基酸(aa),有亮氨酸拉链结构域与螺旋-环-螺旋(HLH)结构域。实验表达和纯化了GF-1-C-Myc蛋白,并制备其多克隆抗体。采用实时定量PCR技术(qRT-PCR)与免疫印迹法(WB)检测了GF-1-C-Myc基因的表达和神经坏死病毒的复制。结果显示,缺乏谷氨酰胺会同时抑制GF-1-C-Myc基因的表达和神经坏死病毒(NNV)的复制,添加谷氨酰胺可同时促进GF-1-C-Myc的表达和NNV的复制;此外,NNV感染可上调GF-1-C-Myc基因的表达,并显著消耗GF-1细胞培养液中的谷氨酰胺。研究表明,GF-1-C-Myc基因可调控宿主谷氨酰胺代谢,从而有利于神经坏死病毒的复制。本结果为防控NNV的感染提供了参考。     

Thyroxine-induced alterations in the testis and seminal vesicles of air-breathing catfish, Clarias gariepinus

Effect of experimentally induced thyroxine overdose on the testis and seminal vesicles was studied in the air-breathing catfish, Clarias gariepinus during the preparatory and the pre-spawning phase. The present study revealed a marked reduction in testosterone level in serum, testis and seminal vesicles (SV). Histological examination showed a considerable reduction in the number of spermatozoa/spermatids in the seminiferous tubular lumen as well as depletion of fluid in the loculi of SV. SDS-PAGE analysis of SV fluid proteins demonstrated a significant decrease in the level of a ~27 kDa protein in thyroxine treated fishes. Evidences are presented here to indicate that thyroid hormone plays a role in regulating testis and SV function in catfish. T.N. Jacob and J.P. Pandey contributed equally     

红色荧光蛋白基因在转基因唐鱼中遗传和表达的稳定性分析

为了评估转红色荧光蛋白(red fluorescent protein,RFP)基因唐鱼新品系的稳定性,研究了RFP基因在不同世代转基因唐鱼中的遗传和表达情况。荧光显微镜下观察显示,RFP基因在F6和F10代所检测的组织器官中均有表达,并且两个世代间相同组织部位的表达水平相似。F6和F10代个体分别配对繁殖实验表明,RFP基因在转基因唐鱼后代中的遗传仍然符合孟德尔分离规律,而且培育出的转基因个体表型特征无显著差异。利用PCR技术在F2、F6和F10代转基因唐鱼基因组中扩增外源性肌球蛋白轻链2启动子、RFP基因编码区和整合位点上下游侧翼区域(片段总长度为4 883 bp),测序结果显示,3个世代间的外源基因序列完全相同,没有发生碱基缺失或突变等现象。研究表明,红色荧光蛋白基因在转基因唐鱼传代培育过程中保持了稳定的遗传和表达。     

Active metabolism in larval and juvenile fish: ontogenetic changes,effect of water temperature and fasting

Oxygen consumption, ammonia excretion and fish swimming speed were measured in fish induced to swim by optomotor reaction in a circular metabolism chamber. The relationship between the swimming speed and fish metabolism described by exponential equations allowed the extrapolation to the standard metabolism, i.e. at zero swimming speed. The partitioning of the catabolised protein in the energy supply was estimated based on AQ (volume of ammonia/ volume of oxygen) values. Weight specific standard metabolism, as expressed by the ammonia excretion rate, decreased by one order of magnitude in coregonids as the fish grew from 20 to 780 mg body weight. The slope of the relationship between oxygen uptake and swimming speed decreased in coregonid ontogenesis. In salmon, after 12 days of fasting 28% of energy used was derived from protein, whilst coregonid juveniles utilized mostly lipid. Active swimming in fasted juveniles of coregonid, as well as in salmon, led to the accelerated utilization of protein as a source of energy, based on AQ coefficients. In juveniles acclimated to a range of water temperatures from 14 to 26°C, the changes in standard or active metabolic rate (expressed as oxygen uptake or ammonia excretion) were described by Q₁₀ coefficients. They were generally higher for the ammonia excretion rate than for the oxygen uptake rate and for active metabolism than for standard metabolism. Utilization of protein as energy for swimming differed significantly between the species, being in general one order of magnitude higher in coregonids than in salmon. The use of protein for swimming activity tended to decrease during coregonid ontogenesis.     

日本黄姑鱼血细胞发生的观察

用Wright’sGiemsa混合染料对日本黄姑鱼外周血涂片以及头肾、肾脏、肝脏和脾脏组织印片染色,观察其中各种血细胞的大小、形态特征,研究了日本黄姑鱼血细胞的发生发育模式。观察发现,在外周血涂片中,除红细胞外,还观察到多种白细胞,主要有单核细胞、嗜中性细胞、嗜酸性细胞和淋巴细胞,而嗜碱性细胞没有被发现。红细胞、淋巴细胞和单核细胞的发生主要在肾脏和头肾,其次是脾脏;粒细胞的发生主要在头肾和脾脏。红细胞的发育经历3个阶段:原红细胞、幼红细胞和成熟红细胞阶段。红细胞在成熟过程中,细胞核的体积逐渐变小,细胞与细胞核的体积比由大变小然后再变大。成熟红细胞除了由幼稚细胞发育而来以外,还可以进行一分为二的方式进行分裂;粒细胞的发育经历5个阶段:原粒细胞、早幼粒细胞、中幼粒细胞、晚幼粒细胞和成熟粒细胞;淋巴细胞经历了3个阶段:原淋巴细胞、幼淋巴细胞和成熟淋巴细胞;单核细胞的发育和淋巴细胞相似,也经历了3个阶段:原单核细胞、幼单核细胞和成熟单核细胞。     

Growth and feed utilisation of rainbow trout subjected to changes in feed lipid concentrations

Rainbow trout Oncorhynchus mykiss grew from 44 to 326 g in 96days when held at 12 °C. Fish were fed to satiation twice dailywith either high (L₁: 30.8%, L₂:31.4%) or lower-lipid feeds (C₁: 18.8%,C₂: 21.8%). Four feeding treatments were studied.Group C₁C₂ received feed C₁ for 43 days(days 0–43) and C₂ thereafter (days 44–96).Groups L₁L₂, L₁C₂ andC₁L₂ were subjected to dietary changes asindicated by the feed designations. After a short period of feedadaptation, fish ingested similar amounts of feed energy i.e., they ateless by weight of the lipid-rich (L) feeds. Feed lipid content did notaffect growth but fish fed L-feed had reduced feed conversion ratio(FCR) compared to fish fed C-feed (0.731 vs. 0.773) during days0–43 (P < 0.01). After 96 days,L₁L₂-fish were lower in body protein(15.8%) than the C₁C₂-fish (16.8%)(P < 0.01). L-feeds also tended to increase percentage lipidand reduce percentage whole body moisture and ash. A higher net proteinutilisation (NPU) was recorded in fish fed L-feeds (43.6%)compared to fish fed C-feeds (38.8%) in days 0–43(P < 0.05). This seemed to be the result of a lower proteinintake rather than a protein-sparing effect of feed lipid. Above athreshold value of approximately 6.5 mg protein eaten·g bodywt_{minus 1}·day_–1, NPU decreased.     

Tissue concentrations of RNA and protein for juvenile brook trout (Salvelinus fontinalis): lagged responses to fluctuations in food availability

To be useful as short-term indices of nutritional status when food availability varies, wet weight-at-length and tissue concentrations of nucleic acids and protein must covary closely with a fish's recent feeding history. We measured changes in these indices for young brook trout (Salvelinus fontinalis) (fork length: 20–34 mm) reared under alternating, 4 to 5 d periods of food provisioning and food deprivation. Weight-at-length corresponded closely with current feeding conditions, being higher when the trout were fed than when they were deprived. Concentrations of RNA and protein, however, did not correspond closely with current feeding conditions. Instead, there were significant carry-over effects whereby responses to feeding conditions experienced in one treatment period were not exhibited until the following treatment period. Food provisioning had positive carry-over effects on concentrations of RNA and protein while food deprivation had negative carry-over effects. Consequently, food-deprived trout sometimes had higher concentrations of RNA and protein than well fed trout. Since wild fish may experience short-term fluctuations in food availability, lagged responses in concentrations of nucleic acids or protein like those reported here could hamper attempts to use these biochemical measures to assess the nutritional status of juvenile fish in the field.Author to whom all correspondence should be addressed.     

Seasonal morphological and biochemical changes of Dahlgren cells implies a potential role of the caudal neurosecretory system (CNSS) in the reproduction cycle of teleostean fish

The purpose of this mini-review is to summarize recent research on the seasonal morphological and biochemical changes of Dahlgren cells in the caudal neurosecretory system (CNSS) of the freshwater teleosts carp Carassius auratus. The quantitative proof for these seasonal changes in the morphology and biochemistry of Dahlgren cells reflects the relationship between the CNSS and the reproduction cycle of fish and implies that the CNSS is probably involved in the reproduction process of fish.     

Effects of experimental infection by Caligus rogercresseyi (Copepoda: Caligidae) on the common jollytail, Galaxias maculatus (Osmeriforme: Galaxiidae) (Jenyns, 1842) in Chile

Galaxias maculatus, the common jollytail, is a native smelt fish with a lacustrine or diadromous life cycle. In Chile, the rearing cycle of this fish includes a freshwater and a marine phase. Several diseases and parasites reported for the freshwater phase could be avoided by rearing the fish in salty or brackish water. Nonetheless, this alternative could result in Caligus rogercresseyi infections. The objective of this study was to evaluate experimentally the capacity of C. rogercresseyi to infect the common jollytail and the effects this parasite may cause to the fish. Prevalence and intensity of experimental C. rogercresseyi infections on G. maculatus were estimated and the effects of this parasitosis on the fish were evaluated on fish survival, weight loss, swimming behavior, and skin damage. Two experiments were carried out with adult fish taken from the Maullín Estuary. Fish were acclimatized to a salinity of 32 ppt and divided into two groups of 20 fish each. The experimental group was infected with copepodid stages of C. rogercresseyi and the other was used as a control. The fish were kept in individual tanks until observing ovigerous female copepods. C. rogercresseyi recognizes G. maculatus as a host under experimental conditions and optimal salinity for the parasite. Results show that under experimental condition copepodid stage can successfully infect the fish, developing to the adult stage, mating, and producing eggs (females). C. rogercresseyi caused alterations in fish behavior, superficial body damage, weight loss, and mortality. Rearing the common jollytail in salty water should take into consideration measures to control these infections as they can damage fish and affect surrounding wild fish populations. There are no records of this parasite in natural common jollytail populations; however, our understanding of the ecological role played by the common jollytail in C. rogercresseyi transmissions among sympatric host species could be greatly improved through further research related to host preferences and epidemiological data for different sympatric host species.