Activation of brain steroidogenesis and neurogenesis during the gonadal differentiation in protandrous black porgy,Acanthopagrus schlegelii

The early brain development, at the time of gonadal differentiation was investigated using a protandrous teleost, black porgy. This natural model of monosex juvenile fish avoids the potential complexity of sexual dimorphism. Brain neurogenesis was evaluated by histological analyses of the diencephalon, at the time of testicular differentiation (in fish between 90 and 150 days after hatching). Increases in the number of both Nissl‐stained total brain cells, and Pcna‐immunostained proliferative brain cells were observed in specific area of the diencephalon, such as ventromedialis thalami and posterior preoptic area, revealing brain cell proliferation. qPCR analyses showed significantly higher expression of the radial glial cell marker blbp and neuron marker bdnf. Strong immunohistochemical staining of Blbp and extended cellular projections were observed. A peak expression of aromatase (cyp19a1b), as well as an increase in estradiol (E₂) content were also detected in the early brain. These data demonstrate that during gonadal differentiation, the early brain exhibits increased E₂ synthesis, cell proliferation, and neurogenesis. To investigate the role of E₂ in early brain, undifferentiated fish were treated with E₂ or aromatase inhibitor (AI). E₂ treatment upregulated brain cyp19a1b and blbp expression, and enhanced brain cell proliferation. Conversely, AI reduced brain cell proliferation. Castration experiment did not influence the brain gene expression patterns and the brain cell number. Our data clearly support E₂ biosynthesis in the early brain, and that brain E₂ induces neurogenesis. These peak activity patterns in the early brain occur at the time of gonad differentiation but are independent of the gonads. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 121–136, 2016     

Oocyte maturation-inducing steroids in protandrous black porgy, Acanthopagrus schlegeli

The study objectives aimed to investigate the maturation-inducing steroid (MIS) in marine protandrous black porgy, Acanthopagrus schlegeli. The characteristics of oocyte maturation were also described. Females were injected with two successive doses of LHRH analog (LHRH-A, 10 and 50 microg/kg of fish). The ovarian tissue was obtained at 6-h intervals for in vitro oocyte maturation. Both 17,20 beta-dihydroxy-4-pregnen-3-one (DHP) and 17,20 beta,21-trihydorxy-4-pregnen-3-one (20 beta-S) were the most effective steroids to induce in vitro maturation (e.g. germinal vesicle breakdown, GVBD) in oocytes cultured for either 24 h or 1 min. 20 beta-S had a better potency than DHP in inducing oocyte maturation. 17-hydroxyprogesterone, 11-deoxycortisol, and 20 beta-21-dihydroxy-4-pregnen-3-one also significantly induced oocyte maturation at high concentrations. The process of oocyte maturation (after the injection of LHRH analog) was founded to be divided into four stages: hormone-insensitive stage (insensitive to gonadotropin and MIS); MIS-insensitive (respond to gonadotropin, but not MIS); MIS-sensitive (respond to MIS); and spontaneous stage (GVBD in the hormone-free condition), respectively. Cycloheximide blocked GVBD at the MIS-insensitive stage, control (hormone-free), and hormone-induced GVBD at the MIS-sensitive stage in a dose-dependent effect.     

Stimulation of testicular function by exogenous testosterone in male protandrous black porgy, Acanthopagrus schlegeli

When 4 mg of testosterone (T) per kg food was given to 1-year-old protandrous male black porgy Acanthopagrus schlegeli for 7 months, gonadosomatic index was significantly higher than when the dose was 0.5 mg kg⁻¹ food. Both doses of T prolonged the spawning season, and increased the number of spermiating fish and milt volume. Sperm concentrations were similar in spermiating black porgy from the treated and control groups. Low levels of oestradiol-17β were observed during the experimental period while elevated levels of plasma T were observed only in March in both control and T-treated groups. Significantly higher levels of plasma 11-ketotestosterone (11-KT) were observed in the 0.5- and 4.0-mg T-treated groups during the spawning season as compared to the control group. The present data suggest that both 0.5- and 4·0-mg T doses stimulate testicular weight, increase numbers of spermiating males and milt volume without affecting the sperm concentrations. Plasma 11-KT concentrations were elevated during T treatment and closely correlated with testicular development and spermiation.     

Regulation of plasma gonadotropin II secretion by sex steroids, aromatase inhibitors, and antiestrogens in the protandrous black porgy, Acanthopagrus schlegeli Bleeker

Plasma gonadotropin II (GTH II) concentrations were significantly higher (approx. 15-20-fold) in estradiol-17beta (E(2)) treated (1.0 microg or 2.5 microg g(-1) body weight) female black porgy from days 4 to 12 compared with the control. E(2) (1 microg g(-1) wt.) had a stronger stimulation on plasma GTH II in early recrudescent phase (low GSI) males (11-fold) than in high GSI and late spermiating males (2.6-fold, P< 0.05). No effect of androgens (testosterone, T; 5alpha-dihydrotestosterone, DHT) on plasma GTH II levels was observed either sex. The levels of plasma GTH II were stimulated in 1,4,6-androstatriene-3,17-dione (ATD, 1 microg g(-1), 2 microg g(-1) body wt.) and fadrozole-treated (1 microg g(-1), 3 microg g(-1) body wt.) groups compared to control. Tamoxifen (1 microg g(-1), 3 microg g(-1) body wt.) but not enclomiphene could stimulate high GTH II levels in plasma. In another experiment of ATD in combination with T, T treatment further attenuated the ATD stimulation of plasma GTH II levels. We concluded that GTH II secretion is positively regulated by an estrogen-specific effect in female and male black porgy. Gonadal stage had significant effects on the responsiveness of GTH II to E(2) stimulation in males. A negative aromatase-dependent feedback control of plasma GTH II levels was also suggested in the protandrous black porgy, Acanthopagrus schlegeli.     

Spermatogenesis in the black porgy,Acanthopagrus schlegeli (teleostei: Perciformes: Sparidae)

The ultrastructure of spermatogenesis and of the spermatozoon of Acanthopagrus schlegeli (Sparidae) are described. The testis is of the unrestricted type. Germ cells are surrounded by cyst cells. Spermiogenesis involves conspicuous modifications such as intracellular movements (diplosome and mitochondria migration, nuclear rotation, and depression) and structural changes (chromatin condensation, shape of mitochondria, and loss of cytoplasm). The mature spermatozoon has a spherical nucleus with a deep, axial nuclear fossa, and an unusual notch, shaped like a bow tie. The short midpiece contains four spherical mitochondria and encircles the basal body of the flagellum. It is concluded that the A. schlegeli spermatozoon is of a primitive type, but that it is characterized by a unique feature which may provide a useful systematic character. © 1993 Wiley-Liss, Inc.     

Aromatase inhibitors block natural sex change and induce male function in the protandrous black porgy,Acanthopagrus schlegeli Bleeker: possible mechanism of natural sex change

The objectives of the present study were to investigate the effects of oral administration of aromatase inhibitors on sex change, milt volume, 11-ketotestosterone (11-KT), and LH in plasma; aromatase activity in gonad, pituitary, and brain in the protandrous fish, black porgy (Acanthopagus schlegeli Bleeker). Two-year-old functional male black porgy were divided into two groups; one was fed a control diet and the other was fed a diet mixed with aromatase inhibitors (AIs; fadrozole and 1,4,6-androstatriene-3,17-dione, each 10 mg/kg feed) for 8.5 mo. A significantly higher gonadosomatic index was observed in the AI group. Fish treated with AIs showed complete suppression of natural sex change. Significantly higher levels of plasma 11-KT, LH, and milt volume were shown in the AI group than the controls. Lower aromatase activity in the gonad, pituitary, forebrain, midbrain, and hindbrain in concordance with the suppression of sex change was observed in the AI group. The data show that aromatase is directly involved in the mechanism of natural sex change of protandrous black porgy. AIs also enhanced male function in concordance with the elevated plasma levels of 11-KT and spermiation in milt volume.     

Characterization of estrogen receptor beta2 and expression of the estrogen receptor subtypes alpha, beta1, and beta2 in the protandrous black porgy (Acanthopagrus schlegeli) during the sex change process

Estrogens play an important role in many physiological processes in both female and male vertebrates, mediated by specific nuclear receptor, estrogen receptors (ERs). We have isolated a third ER (ERbeta2), which was found to contain 2004 nucleotides including an open reading frame that encodes 667 amino acids. We have also cloned ERalpha and ERbeta1 from the published information (GenBank accession nos. AY074780 and AY074779) and investigated the expression pattern of these ER subtypes in the gonads during gonad sex change of black porgy by quantitative polymerase chain reaction. Maturity stages can be divided into five stages during the sex change process from immature male to female (immature male, mature male, male of mostly testis, male of mostly ovary and mature female). The expression of ERalpha mRNA was highest in the ovary of mature female, followed by the testis of mature male and testicular portion of mostly testis. ERbeta1 expression was higher in the mature testis and ovary than in the gonads of other maturity stages. In contrast to that, ERbeta2 was highest in the ovary of mature female, and significantly lower levels of ERbeta2 expression were observed in the gonads of the other maturity stages. The present study describes the molecular characterization of ERbeta2, and documents the expression changes of three ER subtypes during sex change process of the protandrous black porgy.     

Sex-related genes expression in juveniles of red abalone,Haliotis rufescens (Swanson, 1822)

The red abalone, Haliotis rufescens (Swanson 1822), is an important species for commercial aquaculture in Mexico, with annual production levels around 68 t. Not surprisingly, there is great interest in increasing production and its cultivation success. In order to have a better understanding of the genes involved in the sexual differentiation, this study analyzed the early differential expression of eight sex-related genes (VCP 2.2, VERL, VTGI, DMRT1, FP, LYS, SARIP, TEKT A1) by RT-qPCR in abalones from 5 to 45 mm of shell length. Sex identification was evaluated using VERL and LYS genes expression levels. These genes differentiated females and males from 16 mm of shell length and above. All eight sex-specific genes showed expression in all organisms. However, their level was higher in the sex group to which they correspond. In contrast, differential expression levels occurred in individuals with a shell length of 26–35 mm and 36–45 mm. These results suggest that sex gene expression is not entirely sex specific, and sexual differentiation in red abalone occurs between 16 and 25 mm of shell length. More studies are needed to determine the function of these genes in the sex that they are not associated with.     

鲤脑组织低温差异表达候选基因的筛选

采用双标准曲线相对实时荧光定量PCR法,分别以23℃常温对照组和6℃低温待测组的黑龙江鲤脑组织cDNA为模板,以18S rRNA为内参基因,检测26个候选基因的相对表达量。实验数据经显著性分析发现,有5个候选基因在低温条件下表达量显著上升(P<0.01),与对照组相比它们的表达量分别上升了2.11倍、13.9倍、2.52倍、7.38倍和1.83倍,基因功能比对结果表明其编码蛋白产物分别是脂肪酸链延伸蛋白、酰基辅酶A脱氢酶、转录起始因子IIB、肌醇-1-磷酸合成酶、血脑屏障HT7抗原;有7个候选基因在低温下表达量分别下降了21.8%、25.9%、16.6%、23.7%、15.8%、16.3%、42.5%,但对照组和待测组差异不显著(P>0.05),基因功能比对发现它们主要参与抑制糖酵解,促进细胞凋亡和干扰神经系统的重塑活动。上述低温下表达量显著上升的5个冷诱导候选基因的获得为今后进行不耐低温鱼类的基因工程育种提供了基因元件。     

Sexually dimorphic expression and estradiol mediated up‐regulation of a sex‐linked ribosomal gene,RPS6, in the zebra finch brain

Sex‐linked genes are considered to be a major contributor to neural sex differences in zebra finches. While several candidates have been identified, additional ones are continuously being discovered. Here we report on a novel Z‐linked ribosomal gene (rpS6) that is enhanced in the male brain as compared to the female's throughout life. In both sexes, expression of rpS6 is highest at P3 and P8 (just before the onset of morphologically detectable sex differences), decreases around P15, and then remains decreased through adulthood. Analysis of rpS6 mRNA revealed widespread distribution throughout the brain. However, within song regions HVC and RA, mRNA containing cells were greater in males as compared to females. Hormones are also involved in the development of neural dimorphisms, so we additionally investigated whether rpS6 might interact with estradiol (E₂). An up‐regulation of rpS6 gene was observed in both sexes following treatment with E₂ and the effect was approximately twice as large in males as compared with females. These data suggest that rpS6 may be involved in sexual differentiation of the zebra finch brain, and that the effect is facilitated by E₂. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 599–608, 2013     

Age‐related ultrastructural changes of the basement membrane in the mouse blood‐brain barrier

The blood‐brain barrier (BBB) is essential for a functional neurovascular unit. Most studies focused on the cells forming the BBB, but very few studied the basement membrane (BM) of brain capillaries in ageing. We used transmission electron microscopy and electron tomography to investigate the BM of the BBB in ageing C57BL/6J mice. The thickness of the BM of the BBB from 24‐month‐old mice was double as compared with that of 6‐month‐old mice (107 nm vs 56 nm). The aged BBB showed lipid droplets gathering within the BM which further increased its thickness (up to 572 nm) and altered its structure. The lipids appeared to accumulate toward the glial side of the BM. Electron tomography showed that the lipid‐rich BM regions are located in small pockets formed by the end‐feet of astrocytes. These findings suggest an imbalance of the lipid metabolism and that may precede the structural alteration of the BM. These alterations may favour the accretion of abnormal proteins that lead to neurodegeneration in ageing. These findings warrant further investigation of the BM of brain capillaries and of adjoining cells as potential targets for future therapies.