Seabream GnRH immunoreactivity in brain and pituitary of XX and XY Nile tilapia, Oreochromis niloticus during early development

Seabream gonadotropin-releasing hormone (sbGnRH)-the chief preoptic area-hypothalamus (POA-H) form of GnRH in tilapia is involved in sexual maturation. In this study, we investigated the qualitative changes in ontogeny of sbGnRH immunoreactivity (ir-), between sexes to understand its impending role during sex differentiation. For this, the differences in immunocytochemical localization of sbGnRH in genetically male (XY) and female (XX) fish were studied from 1 day after hatching (dah), through the critical period of sex differentiation (7-21 dah) to 40 dah and mature Nile tilapia. Specific antisera against sbGnRH were used for immunolocalization. SbGnRH ir- neurons were observed in POA-H as early as 5 and 15 dah in XY fish and XX fish, respectively. Higher ir- was detected in the POA-H of XY tilapia compared with XX population till 10 dah. There was a qualitative drop in sbGnRH ir- neurons/cell bodies in POA-H around 20 dah till 30 dah in XY population compared with other durations. SbGnRH ir- cells were detected in pituitary of XX fish by 15 dah and in XY fish around 10 dah but seemed to drop down by 20 dah in XY whereas it continued to remain steady in XX fish. The sbGnRH ir- in XY fish showed a rise from 35 dah and thence till 40 dah. This study revealed subtle differences in POA-H and pituitary sbGnRH ir- during early development between genetic male and female fish with possible implications in sex differentiation.     

Dynamics and histological characteristics of gonadal sex differentiation in pejerrey (Odontesthes bonariensis) at feminizing and masculinizing temperatures

This study evaluated the effects of different temperatures on the histological process of sex differentiation in the pejerrey Odontesthes bonariensis, a fish with marked temperature-dependent sex determination (TSD), at feminizing, neutral, and masculinizing temperatures. Fish reared at three temperatures (17 degrees C, 24 degrees C, and 29 degrees C) from hatching were sampled weekly until 11 weeks and their gonads were examined by histology. The percentages of females at 17 degrees C, 24 degrees C, and 29 degrees C were 100%, 73%, and 0%, respectively. Sex differentiation occurred earlier and at a smaller body size at higher temperatures in both sexes. The first signs of ovarian differentiation were observed at 4 and 7 weeks at 24 degrees C and 17 degrees C, respectively, and those of testicular differentiation at 4 and 7 weeks at 29 degrees C and 24 degrees C, respectively. Body or gonadal growth rates before sex differentiation were not proportional to temperature and showed no sexual dimorphism at 24 degrees C, where both sexes were present. Thus, differential growth rate is probably not a factor in TSD or histological sex differentiation in pejerrey. Blood vessels were formed before sex differentiation in both sexes and at all temperatures, and may be important for sex differentiation. No signs of intersexuality were found in any of the groups, and this characterizes pejerrey as the differentiated type of gonochorist even at feminizing and masculinizing temperatures. Ovaries were formed by the same histological processes at feminizing (17 degrees C) and neutral (24 degrees C) temperatures and without any pathological features such as germ cell degeneration. The process of testicular formation was generally similar at 24 degrees C and 29 degrees C, but some fish at 29 degrees C had widespread germ cell degeneration before sex differentiation. This suggests that pathological processes leading to germ cell death, such as heat-induced dysfunction of the supporting somatic cells, could be involved in masculinization of the genetic females at high temperatures.     

南方鲶性腺分化的组织学观察

用芳香化酶抑制剂(Fadrozole)、雌激素受体拮抗剂(Tamoxifen)对人工孵化的南方鲶(Silurus meridionalis)幼鱼进行雄性化诱导处理(口服),获得雄鱼。对孵化后第5—130d的南方鲶幼鱼性腺进行组织学观察,结果表明,在实验条件下,南方鲶性腺分化发生在孵化后7d左右,雌雄性分化过程差异明显。雌鱼卵巢腔在孵化后12d左右形成,生殖细胞在孵化后35d左右快速增殖,成熟分裂最早发生在孵化后55d左右;雄鱼生殖细胞在孵化后130d左右快速增殖,成熟分裂最早发生在孵化后130d左右。雌性性腺分化早于雄性。     

In vivo fertilization and development of medaka eggs initiated by artificial insemination

Fertilization and development in the ovarian cavity of oviparous fish, Oryzias latipes, were examined using the S-rR strain in which the sex genotype can be easily distinguished by the body color of the fish. Mature eggs were fertilized within the ovarian cavity after a sperm suspension was artificially introduced with a small bore-pipette through the urinogenital opening. Three batches of eggs ovulated within 48 hrs were fertilized and began to develop in the ovarian cavity, while eggs ovulated 72 hrs post-insemination (PI) were no longer fertilized. These observations indicate that ovulation occurs irrespective of the existence of developing embryos within the ovarian cavity. All embryos developing in the ovarian cavity were, however, retarded and ceased development before the stage of initiation of blood circulation at room temperature. These embryos developed normally and hatched after they were transferred from the ovarian cavity into regular saline 48 or 72 hrs PI. When these individuals matured sexually, their sex differentiation was found to be normal, and sex reversal was not observed.     

Where does gonadal sex differentiation begin? Gradient of histological sex differentiation in the gonads of pejerrey,Odontesthes bonariensis (Pisces,Atherinidae)

This study investigated the possibility that the histological process of gonadal sex differentiation in pejerrey (Odontesthes bonariensis), a fish with marked temperature-dependent sex determination (TSD), occurs through a predictable gradient of differentiation as opposed to simultaneous or random differentiation throughout the gonad. For this purpose, fish reared at 17 degrees, 24 degrees, and 29 degrees C from hatching were sampled weekly for 11 weeks, fixed, and prepared for histological observation of serial cross-sections of the gonads. The thermal manipulation and sampling procedure ensured the availability of males and females at various degrees of gonadal sex differentiation. The location of the differentiated area(s) was estimated in the right and left gonads of 17 females and 14 males selected among the available specimens so as to represent increasing degrees of differentiation. The analysis revealed that sex differentiation followed a gradient from the anterior to posterior areas of the gonads regardless of sex. Furthermore, plotting of the degree of sex differentiation in the right gonad as a function of the degree of differentiation of the left gonad clearly showed that sex differentiation only begins in the right gonad when 10-30% of the length of the left gonad has already differentiated. The mean rostral edge of the differentiated areas in females was 9% and 10.8% for the left and right gonads, respectively, while for males these values were 7.3% and 7.0%, respectively. Thus, it was established that ovarian and testicular differentiation in pejerrey follow both a cephalocaudal and a left-to-right gradient. Possible explanations for this gradient and its relevance for TSD in pejerrey, that is, as a mechanism to prevent discrepant differentiation of male and female features within the same gonad, are discussed.     

Effect of rearing temperature on sex ratio in juvenile Atlantic halibut, Hippoglossus hippoglossus

Several flatfish species exhibit temperature-dependent sex determination. This research investigated the effects of rearing temperature on sex ratio in Atlantic halibut, Hippoglossus hippoglossus, a species in which females grow larger and faster than males under culture conditions. Previous research has shown that ovarian differentiation occurs in Atlantic halibut in the size interval of 38–50 mm, and precedes the differentiation of testes. In the current study, triplicate groups of juvenile Atlantic halibut were reared at each of three temperatures (7, 12 and 15°C) from an initial mean size of 21 mm to a final mean size of 80 mm (total length). The sex of each fish was then determined by macroscopic and histological examination of the gonads. Sex ratios were not significantly different from 1:1 in any group, suggesting that sex in this species is not influenced by temperature.     

Gonadal sex differentiation and effect of rearing temperature on sex ratio in black rockfish (Sebastes schlegeli)

Temperature-dependent sex determination has been demonstrated in some species of fish, and a high temperature during the period of sex differentiation typically produces a male-dominant population. This research investigated the gonadal sex differentiation and effect of rearing temperature on the sex ratio in larval black rockfish Sebastes schlegeli, which is a viviparous species. Two types of gonads were histologically distinguishable in fish 20 mm in total length (TL). The putative ovary started forming an ovarian cavity, while the putative testis was not clearly differentiated until 51 mm TL. In a temperature-controlled experiment, the proportions of females were 45% at 10°C, 46% at 14°C, 50% at 18°C, 63% at 22°C, and 83% (significantly different from 1:1 sex ratio) at 24°C. These results suggest that morphological sex differentiation in black rockfish occurs at approximately 20 mm in TL, and it is possible that high temperatures (24°C) induce not a male- but a female-dominant population in this species.     

Determination of sex and maturity stage of great sturgeon (Huso huso) using steroid sex hormones profiles,histology and laparoscopic images of gonads

We studied sex and maturity stage of great sturgeon with different reproductive stages using sex steroid hormones concentrations in blood plasma, gonad histology and laparoscopic technique. One hundred sixty farmed great sturgeons at ages between 1 and +12 years and weight ranged from 0.45 to 49.65 kg (80 males and 80 females, 20 fish of each maturity stage) were selected. Mean sex steroid levels showed that testosterone (T) and 11ketotestosterone (11KT) in plasma varied significantly by sex and maturity stage. But the mean concentrations of 17B‐estradiol (E2) varied significantly only between the maturity stages I and III. All fish were sex determined by laparoscopy and histology examinations confirmed it. Our results showed, however laparoscopy was effective method for sex determination at four maturity stages, but young fish at maturity stage I required gonadal histology examinations to confirm sex. The present study results showed that some steroid sex hormones (T and 11KT) can be used to determine sex and reproductive stage in great sturgeon, especially in later maturity stages. Although the laparoscopy may be stressful and cause small scars on the body of the fish, but it is a fast, effective, and cheap method for sex and maturity stage assessment (30 to 40 s for each fish), especially at later maturity stages. Histology of gonads is a very accurate, but it is an invasive method and not a suitable technique for commercial purposes. Histology is more suited for scientific studies than commercial application. The ability to use minimally invasive methods for sex determination will assist broodstock management and conservation in sturgeons.     

Monogamous mating system and sexuality in the gobiid fish, <Emphasis Type="Italic">Trimma marinae</Emphasis> (Actinopterygii: Gobiidae)

The mating system and sexuality of the gobiid fish Trimma marinae were investigated in aquaria and by gonadal histological examination. The male to female sex ratio in the study aggregation was female biased (14:27), and females were larger than males. T. marinae were monogamous because they established continuous pairs and spawned repeatedly with the same individuals. Observations of aggressive behavior suggested that the monogamous mating system resulted from female mate guarding. We also performed a rearing experiment to test whether sex change occurs in this species. None of the males or females reared separately in aquaria for 63 days changed sex. Additionally, gonadal histology revealed that mature fish had unisexual gonads (testis or ovary). These results strongly suggest that T. marinae is gonochoristic. However, immature fish had a bisexual gonadal structure, indicating juvenile hermaphroditism.     

Can a minority of informed leaders determine the foraging movements of a fish shoal?

There is no information on whether the daily foraging movements of fish shoals are the result of chance, the collective will of all shoalmates, or the leadership of a few individuals. This study tested the latter possibility. Shoals of 12 golden shiners, Notemigonus crysoleucas, were trained to expect food around midday in one of the brightly lit corners of their tank. They displayed daily food-anticipatory activity by leaving the shady area of their tank and spending more and more time in the food corner up to the normal time of feeding. Past this normal time they remained in the shade, even on test days when no food was delivered. Most of these experienced individuals were then replaced by na?ve ones. The resulting ratio of experienced:na?ve fish could be 5:7, 3:9 or 1:11. On their own, na?ve individuals would normally spend the whole day in the shade, but in all tests the experienced individual(s) were able to entrain these more numerous na?ve fish out of the shade and into the brightly lit food corner at the right time of day. Entrainment was stronger in the 5:7 than in the 1:11 experiment. The test shoals never split up and were always led by the same fish, presumably the experienced individuals. These results indicate that in a strongly gregarious species, such as the golden shiner, a minority of informed individuals can lead a shoal to food, either through social facilitation of foraging movements or by eliciting following behaviour. Copyright 2000 The Association for the Study of Animal Behaviour.     

Expression patterns of runx2, sparc, and bgp during scale regeneration in the goldfish Carassius auratus

Teleost fish scale is a dermal skeleton equipped with a strong regenerative ability. Owing to this regenerative ability, teleost fish scale can be used as a model for the regeneration of the dermal skeleton. However, there is insufficient fundamental knowledge of the regeneration, and this limits the usage of fish scale. In this study, as a first step toward understanding the molecular mechanism of the cellular differentiation during scale regeneration, we cloned the cDNAs for osteoblast-related proteins (Runx2, Sparc, and Bgp) in goldfish, and analyzed their expressions during scale regeneration. The expression profiles of these genes during scale regeneration were similar to those during mammalian osteoblastic differentiation. Specifically, runx2 expression was increased at the earliest time point, followed by sparc expression and then bgp expression. In the earlier stages, these genes were expressed in cells that formed cellular condensations and the flat cells surrounding them in the scale pocket. As the regeneration proceeded, the expressions became restricted to the episquamal, hyposquamal, and marginal scleroblasts and the cells around the marginal area of the regenerating scale. These results strongly suggest that (1) the differentiation mechanism of scleroblasts is similar to that of mammalian osteoblasts and odontoblasts, (2) scleroblast differentiation occurs around the cellular condensations at the early regeneration stage and is restricted to the marginal area of the scale at the later stage, and (3) the differentiation mechanisms are similar between the episquamal scleroblasts that produce the external layer and the hyposquamal scleroblasts that produce the basal plate.     

Superficial cell differentiation during embryonic and postnatal development of mouse urothelium

After drastic urothelial destruction around birth and around postnatal day 6, mouse urothelial renewal starts each time de novo. The differentiation of superficial cells during urothelial restoration was followed for the first time from embryonic day 15 to postnatal day 6 by the detection of differentiation markers: cytokeratins, uroplakins and apical membrane specialization. The differentiation markers of short-lived superficial cells were studied before and after urothelial destruction. Three distinctive types of superficial cells, typical for certain developmental period, were characterised: cells at low differentiation stage with microvilli and cilia, expressing CK7 and CK18, detected on embryonic day 15; cells at advanced differentiation stage with star-like arrangement of prominent membrane ridges, expressing CK7 and CK20, present between the two urothelial destruction events; highly differentiated cells with typically jagged apical surface, expressing CK7 and CK20, found twice during development. This cell type appears for the first time on embryonic day 18 as the terminal stage of embryonic differentiation. It was found again on postnatal day 6 as an initial stage of differentiation, leading toward terminally differentiated cells of the adult urothelium. Our work proves that apical membrane specialization is the most valuable differentiation marker of superficial cells.     

Early origins of the X and Y chromosomes: lessons from tilapia

Differentiated sex chromosome pairs in diverse species display certain common characteristics, normally comprising one largely heterochromatic genetically inactive chromosome and one euchromatic genetically active chromosome (e.g. the mammalian Y and X respectively). It is widely accepted that dimorphic sex chromosomes evolved from homologous pairs of autosomes. Although the exact mechanisms through which the pair diverged are not fully understood, an initial suppression of recombination in the sex-determining region is required by all of the major theories. Here we address the question of the mechanism by which this initial suppression of recombination occurs. Our model postulates that the stochastic, de novo accumulation of heterochromatin in the sex determining region can delay pairing of the sex chromosomes in meiosis, resulting in a decrease in recombination. Data to support this model is presented from the cichlid fish, Oreochromis niloticus. Although such a decrease would in most circumstances be evolutionarily disadvantageous, if the region concerned included the major sex determining gene and other gene(s) with sex-specific functions, then this would be selectively advantageous and could trigger the process(es) which, ultimately, lead to the differentiation of the sex chromosomes.     

Sex-Linked Inheritance of the lf Locus in the Medaka Fish (Oryzias latipes)

The lf (leucophore free) locus was previously reported autosomal recessive in the medaka fish (Oryzias latipes). However, extensive linkage analyses in this study using various strains revealed that the lf locus was closely sex-linked. The recombination frequency between lf and the male determining factor (y) was 2.2% (10 recombinants out of 464 progeny). Because the lf/lf homozygous fish do not have visible leucophores, they are distinguishable from wild type in early developmental stages. In the Qurt strain with heterozygous sex chromosomes (X(lf)/X(lf) in females and X(lf)/Y(+) in males), we can predict sex of each embryo on second day after fertilization. The strain should be a very useful material for studying sex determination or differentiation mechanisms in the medaka fish.     

Zebrafish monosex population reveals female dominance in sex determination and earliest events of gonad differentiation

The zebrafish is a popular model for genetic analysis and its sex differentiation has been the focus of attention for breeding purposes. Despite numerous efforts, very little is known about the mechanism of zebrafish sex determination. The lack of discernible sex chromosomes and the difficulty of distinguishing the sex of juvenile fish are two major obstacles that hamper the progress in such studies. To alleviate these problems, we have developed a scheme involving methyltestosterone treatment followed by natural mating to generate fish with predictable sex trait. Female F1 fish that gave rise to all-female offspring were generated. This predictable sex trait enables characterization of gonadal development in juvenile fish by histological examination and gene expression analysis. We found the first sign of zebrafish sex differentiation to be ovarian gonocyte proliferation and differentiation at 10 to 12 days post-fertilization (dpf). Somatic genes were expressed indifferently at 10 to 17 dpf, and then became sexually dimorphic at three weeks. This result indicates clear distinction of male and female gonads derived independently from primordial gonads. We classified the earliest stages of zebrafish sex determination into the initial preparation followed by female germ cell growth, oocyte differentiation, and somatic differentiation. Our genetic selection scheme matches the prediction that female-dominant genetic factors are required to determine zebrafish sex.     

埋植17α-甲基睾酮诱导点带石斑鱼性转化技术

论述了埋植17α-甲基睾酮诱导点带石斑鱼(Epinephelus malabaricus)性转化的方法,包括药物的处理、用量、手术操作等．3龄的雌性点带石斑鱼经过2次埋植外源激素手术转化为功能性雄鱼,139d后58％可挤出精液,301d后100％可挤出精液．功能性雄鱼用于人工繁殖,亲鱼自然产卵。1999～2000年2年内共孵出仔鱼7103万尾．所得仔鱼用于鱼苗培育,得到发育正常的幼鱼．认为埋植外源激素诱导点带石斑鱼完成性转化的方法简便可行,能满足规模化人工繁殖的需要．     

Granulated metrial gland cells in the pregnant uterus of mice expressing the collagen mutation tight-skin (Tsk/+)

Summary Influences of the extracellular matrix (ECM) on the differentiation and distribution of granulated metrial gland (GMG) cells, a uterine natural killer (NK)-like cell subset, were studied by histological examination of implantation sites in the mouse mutant Tsk/+. Tsk/+ mice overproduce collagens I and III. GMG cell differentiation appeared to progress normally in Tsk/+ mice between days 6.5 and 12.5 of gestation. The distribution of GMG cells, however, was abnormal. Significant numbers of GMG cells were found in the antimesometrial and lateral decidual regions at day 8.5 of gestation and in the regions between implantation sites until day 10.5 of gestation. Loss of GMG cells from these regions normally occurs by day 6.5 of gestation. These data suggest that alterations to the ECM change the migration properties or life span of GMG cells.     

Masculinization of the gynogenetic juvenile ship sturgeon (Acipenser nudiventris Lovetsky, 1828) using 17α‐methyl testosterone

In sturgeons, the induction of gynogenesis and sex reversal could be important for potential production of neomale sturgeon and all‐female progeny for caviar production. The aim of this study was sex reversal of ship sturgeon (Acipenser nudiventris Lovetsky, 1828) gynogen into male sex. Five‐month‐old gynogens were sex reversed into male by including 17α‐methyl testosterone in their food for 7 months. Three treatments were considered as follows: (a) without treated (gynogen control), (b) 10 mg MT/kg diet, and (c) 50 mg MT/kg diet. All treatments (60 individuals) were sampled both the 30 and 36 months old and their sex was determined using classical histology method of gonad. The sex ratio of the progenies in the gynogen control were 73.3% female and 26.7% male. In treatment of 10 mg MT/kg feed, 18 specimens were studied that half of them (50%) showed pseudo‐testicular structure in the female gonad. That is 50% of the specimens were intersex, 27.7% were male and 22.3% were female. All of the fish fed by 50 mg MT/kg feed had been sex reversed to male. Sexual maturation of these fish had been recognized in stage III at 36 months old. In conclusion, 50 mg MT/kg feed found as effective dose for successful sex reversal in gynogenetic ship sturgeon.