Molecular cloning, characterization and expression analysis of a novel PDRG1 gene from black tiger shrimp (Penaeus monodon)

Abstract P53 And DNA Damage-Regulated Gene 1 (PDRG1) is a novel gene which plays an important role in chaperone-mediated protein folding. In the present study, the full-length complementary DNA (cDNA) sequence of the PDRG1 gene from Penaeus monodon (PmPDRG1) was cloned by the rapid amplification of cDNA ends (RACE) method. The cDNA of PmPDRG1 spans 1,613 bp, interrupted by only one short intron, and encodes a protein of 136 amino acids with calculated molecular weight of 15.49 kDa. The temporal expression profile of PmPDRG1 in different tissues and in different developmental stages of the ovary was investigated by real-time quantitative PCR (RT-qPCR). An RNA interference (RNAi) experiment was performed to study the relationship between P. monodon p53 (Pmp53) and PmPDRG1, and the results showed that the relative expression level of PmPDRG1 mRNA was notably up-regulated from 12 h to 96 h after Pmp53 was silenced both in ovary and hepatopancreas. To further explore the role of PmPDRG1 in ovarian development, dopamine (DA) and 5-hydroxytryptamine (5-HT)-injected shrimps were analyzed by RT-qPCR, indicating that PmPDRG1 may be involved in the regulation of ovarian development of P. monodon.

Evaluation of apoptosis as a mechanism of follicular cell atresia in the ovaries of cattle (bos indicus) and buffalo (bubalus bubalis) fetuses

The objective of this study was to investigate the occurrence of apoptosis in the ovaries of cattle and buffalo fetuses between 4 and 8 months old by the terminal deoxynucleotidyltransferase - mediated dUTP nick end labeling (TUNEL) assay . Histological analysis of the ovarian t issue showed that apoptosis occurred at all ages evaluated , presenting a similar pattern among different fetal stages in both species . Within species, secondary follicles displayed a higher (P 0.05) of apoptotic follicular cells among the three follicular classes compared . C omparing resul ts between species, secondary follicles had a higher (P < 0.05) mean number of TUN EL positive cells in bovine fetuses; however , this difference was proportional to the larger number of follicular cells present in secondary follicles in this species . In summary, the TUNEL method was effective for the detection of apoptosis in the support ing cells of ovarian follicles from bovine and buffalo fetuses with apoptosis occurring at similar rates in both species between 4 and 8 months of gestational age. Further studies are needed to better understand the dynamics of apoptosis as a regulator of follicular atresia in fetal ovaries from these species, as well as the potential involvement of the oocyte in this process.

Análise do desenvolvimento e das glicoproteínas presentes nos folículos ovarianos de poecilia vivipara (cyprinodontiformes, poeciliidae) / Analyses of the development and glycoproteins present in the ovarian follicles of poecilia vivipara (cyprinodontiformes, poeciliidae)

Os aspectos morfofuncionais da oogênese do Poecilia vivipara foram estudados nesse trabalho. Esse estudo contribuiu para o aprimoramento das informações sobre a biologia reprodutiva e do desenvolvimento de espécies com fecundação interna, em especial aquelas pertencentes à família Poecilidae. Para tanto, caracterizou-se os estágios de maturação gonadal e desenvolvimento folicular através de análises mesoscópicas, histológicas, histoquímicas e citoquímicas com lectinas. O estudo mesoscópico permitiu a classificação do desenvolvimento ovariano em seis estágios: imaturo, em maturação I, em maturação II, maturo I, maturo II e pós-desova. O exame microscópico dos ovários permitiu a identificação dos oócitos pré-vitelogênicos (OPVt) e vitelogênicos (OVt). Na fase pré-vitelogênese, verificou-se os oócitos tipo I (OI) e tipo II (OII), e na fase vitelogênese, foram encontrados oócitos tipo III (OIII), tipo IV (OIV) e os pós-fertilização (OV). Com o avanço da oogênese, o citosol dos oócitos aumentaram em volume, com crescente acúmulo de grânulos citoplasmáticos, caracterizando a vitelogênese. Além disso, a zona radiata (ZR) aumentou de espessura e alterou suas características histocitoquímicas, e o epitélio folicular (EF), inicialmente delgado e constituído por células pavimentosas, nos FOs na fase III tornaram-se cúbico simples. As análises histoquímicas e citoquímicas permitiram diagnosticar alterações na composição molecular das estruturas que formam os FOs ao longo do desenvolvimento gonadal. O presente estudo indica diferenças no sistema reprodutor feminino entre espécies de peixes com fecundação interna e aquelas com fecundação externa e sugere que o P. vivipara pode ser usado como modelo experimental em testes de toxicidade ambiental.

The morphofunctional aspects of oogenesis of Poecilia vivipara were studied aiming to understand the reproductive biology and development of species with internal fertilization, particularly those belonging to the family Poeciliidae. The stages of gonadal maturation and follicular development were characterized using mesoscopic, histological, histochemical, and lectin cytochemical analyses. Through mesoscopic evaluation the ovarian development was classified in six phases of development: immature, in maturation I, in maturation II, mature I, mature II, and post-spawn. Based on microscopic examination of the ovaries, we identified the presence of oocytes types I and II during the previtellogenic phase and types III, IV, and V during the vitellogenic phase. As oogenesis proceeded the oocyte cytosol increased in volume and presented increased cytoplasmic granule accumulation, characterizing vitellogenesis. The zona radiata (ZR) increased in thickness and complexity, and the follicular epithelium, which was initially thin and consisting of pavimentous cells, in type III oocytes exhibited cubic simple cells. The histochemical and cytochemical analyses revealed alterations in the composition of the molecular structures that form the ovarian follicle throughout the gonadal development. Our study demonstrated differences in the female reproductive system among fish species with internal and external fertilization and we suggest P. vivipara can be used as experimental model to test environmental toxicity.

Adult carbohydrate feeding affects reproduction of Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae)

Reproduction of most insects depend on nutrients accumulated during the larval stage, but many lepidopteran species will also depend on nutrients obtained at the adult stage. Feeding at the adult stage allows the intake of carbohydrate and amino acid rich solutions, which may have an effect on the species reproduction and population growth. The objectives of the current study were to characterize the effects of sugar consumption by adults of the potato tuber moth, Phthorimaea operculella (Zeller), on its reproduction. To initially test the food intake by adults, a 10 percent honey solution or water (control), both containing a liquid dye were offered to adult insects 24h after emergence, and the presence of the dye was observed by analysis of their digestive system. The effects of adult feeding on a 10 percent honey solution on the reproductive performance of P. operculella were evaluated by assessing the oviposition rate, fecundity and fertility. Adult feeding was proved by the presence of the dye within the digestive system of adults of P. operculella. Although the oviposition rate and fertility were not affected by adult feeding, female fecundity was higher in honey-fed females as compared to the water-fed females.

Ovarian development and spawning of Serra Spanish mackerel in coastal waters of Northeastern Brazil / Desenvolvimento ovariano e desova da Serra nas águas costeiras do nordeste do Brasil

The Serra Spanish mackerel, Scomberomorus brasiliensis is an important fishery resource of the Atlantic Ocean and is a major component of artisanal fisheries in northern and northeastern Brazil. Ovarian development and spawning period of S. brasiliensis were investigated using both macroscopic and histological techniques. Mean monthly values of GSI and ovarian maturation indicate that the main spawning period occurs during the rainy season.

A Serra, Scomberomorus brasiliensis, é uma espécie marinha de valor comercial do oceano Atlântico sendo um maior componente de pesca artesanal no norte e nordeste brasileiros. Desenvolvimento ovariano e época de desova de S. brasiliensis foram investigados utilizando-se técnicas macroscópicas e histológicas. Valores mensais médios de IGS e maturação gonadal indicam que a época de desova ocorre durante o período de chuva.

Physiological age and longevity of Anopheles (Kerteszia) cruzii Dyar Knab (Diptera: Culicidae) in the Atlantic Forest of Southern Brazil

We analyzed the reproductive status, ovarian development, daily survival rate, and length of the gonotrophic cycle in females of Anopheles ( Kerteszia ) cruzii Dyar & Knab, to determine how these factors influence the risk of malaria transmission in the coastal region of the state of Paraná, southern Brazil. In the Palmito State Forest, Paranaguá, females were captured at dawn and dusk by aspiration, bimonthly from December 2006 through March 2007. A total of 2,268 females were captured, of which 454 were dissected. Of these, 48 percent were parous, 50 percent not reproductive, 73 percent in Christopher and Mer stages I and II, 23 percent in stages III to V, 55 percent nulliparous, 14 percent uniparous, and 11 percent had blood in their midgut. Daily survival was 0.24 ± 0.03 overall, 0.51 ± 0.04 for females captured at dusk, and 0.25 ± 0.03 for those captured at dawn. The Davidson equation for calculation of the gonotrophic cycle was inadequate for An. cruzii populations. Females captured at dusk had a higher survival rate than those from dawn, which means that more females of the dusk population enter the parasite extrinsic cycle. The continuous activity and abundance of A. cruzii in the Palmito State Forest suggests that the conditions are very favorable for its development, with a potential for participation in the protozoan's transmission cycle.

Isolation and characterization of genes functionally involved in ovarian development of the giant tiger shrimp Penaeus monodon by suppression subtractive hybridization (SSH)

Suppression subtractive hybridization (SSH) libraries between cDNA in stages I (previtellogenic) and III (cortical rod) ovaries of the giant tiger shrimp (Penaeus monodon) were established. In all, 452 ESTs were unidirectionally sequenced. Sequence assembly generated 28 contigs and 201 singletons, 109 of which (48.0 percent) corresponding to known sequences previously deposited in GenBank. Several reproduction-related transcripts were identified. The full-length cDNA of anaphase promoting complex subunit 11 (PmAPC11; 600 bp with an ORF of 255 bp corresponding to a polypeptide of 84 amino acids) and selenoprotein M precursor (PmSePM; 904 bp with an ORF of 396 bp corresponding to a polypeptide of 131 amino acids) were characterized and reported for the first time in penaeid shrimp. Semiquantitative RT-PCR revealed that the expression levels of PmSePM and keratinocyte-associated protein 2 significantly diminished throughout ovarian development, whereas Ser/Thr checkpoint kinase 1 (Chk1), DNA replication licensing factor mcm2 and egalitarian were down-regulated in mature ovaries of wild P. monodon (p < 0.05). Accordingly, the expression profiles of PmSePM and keratinocyte-associated protein 2 could be used as biomarkers for evaluating the degree of reproductive maturation in domesticated P. monodon.

Morphological characteristics of ovarian development of two Nile tilapia (Oreochromis niloticus) strains in mixed-culture systems / Características morfológicas do desenvolvimento ovariano de duas linhagens de tilápia-do-nilo (Oreochromis niloticus), em sistemas de cultivo misto

This study was carried out to morphologically characterize and classify the stages of gonad development in different Nile tilapia strains (Oreochromis niloticus). Eighty-four and ninety-two ovaries from Bouaké and Chitralada strains, respectively, were evaluated at different ovarian developmental phases: initial (104 days of culture), intermediate (152 days of culture), and the final (279 days of culture). The ovaries were microscopically evaluated and submitted to histological processing and hematoxylin-eosin staining to determine their characteristics and be classified. No morphological differences in ovaries between strains were observed during the initial phase (stage A - immature). During the intermediate growing phase, higher gonad development was observed for Chitralada strain (stage B - maturation) in comparison with Bouaké strain (stage A - immature). During the final growing phase, no differences between strains were observed for morphological characteristics (stage C - mature). Despite the similarities in reproductive behavior of the Bouaké and Chitralada females at the end of the final growing phase (gain weight phase), differences for macroscopic and microscopic aspects and oocytes during the initial and intermediate growing phases of the strains were observed.

Este trabalho teve como objetivo caracterizar morfologicamente e classificar os estádios de desenvolvimento gonadal de tilápias do Nilo (Oreochromis niloticus) de linhagens distintas. Foram avaliados 84 ovários da linhagem Bouaké e 92 da linhagem Chitralada, em diferentes fases de desenvolvimento: inicial (imatura - 104 dias de cultivo), intermediária (crescimento - 152 dias de cultivo) e final (ganho de peso - 279 dias de cultivo). Os ovários foram analisados macroscopicamente e submetidos a procedimento histológico, corados com hematoxilina-eosina, para determinação das características microscópicas e subsequente classificação. Não foram observadas diferenças morfológicas nos ovários entre as linhagens na fase inicial (estádio A - imaturo). Na fase intermediária, os ovários revelaram diferenças morfológicas, evidenciando maior desenvolvimento gonadal para a linhagem Chitralada, (estádio B - maturação) em relação à Bouaké (estádio A). Na fase final (ganho de peso) os ovários não apresentaram diferenças morfológicas entre as linhagens (estádio C - maduro). Apesar de semelhanças no comportamento reprodutivo das fêmeas das linhagens Bouaké e Chitralada, na fase final (ganho de peso), diferenças, em relação à classificação da fase intermediária nos aspectos macroscópicos e microscópicos e diâmetro dos ovócitos, foram observadas.

Desarrollo ovárico y su relación con las concentraciones séricas de 17β-estradiol y 17α-hidroxi- 4-pregnen-3-ona en hembras de primera maduración de pez blanco, Chirostoma humboldtianum (Atheriniformes: Atherinopsidae) / Association between ovarian development and serum concentrations of 17β-estradiol and 17α-hydroxy-4-pregnen-3-one in first maturation females of the shortfin silverside fish, Chirostoma humboldtianum (Atheriniformes: Atherinopsidae)

The first reproductive cycle of 50 females of the shortfin silverside, Chirostoma humboldtianum (Valenciennes) in culture conditions was analyzed. Ovarian developmental stages, gonadosomatic (GI) and hepatosomatic (HI) indexes were described. Histo-logical description of the ovaries and quantification of estradiol serum levels (E2) and 17α-hydroxy-4-pregnen-3-one (17-P4) by radioinmune assay were performed. Results showed a first reproductive season longer than six months. Females initiated spawning at the age of one year. Four ovarian maturation stages (I to IV) were determined during the spawning season and one (V) during the non-spawning season, the last one showed a non-defined pattern of development. The GI and HI indexes values indicate a positive lineal relation to body-weight only during the reproductive season (r² = 0.74 and r² = 0.86, P < 0.05, respectively). Histological analysis of the ovaries indicated that the species correspond to the pattern described as group-synchronous with multiple spawnings. The follicular population showed reproductive activity during the first stages of maturation with a predominant population of pre-vitellogenic follicles. As the ovarian maturation increased, the presence of all the follicular developmental stages was observed, with a tendency of an increase of vitellogenic and mature follicles. The concentration of circulating sexual steroid hormones of estradiol (E2) and 17α-hydroxy-4-pregnen-3-one (17-P4) were high during stage I (2.5 ± 0.7 ng/m L and 2.4 ± 0.3 ng/mLfor E2 and 17-P4, respectively); a decrease was observed during stage II, and the highest values were observed in stage IV (7.6 ± 2.1 ng/mL for E2 and 1.8 ± 0.9 ng/mL for 17-P4). The same pattern was observed during non-spawning season (ANOVA P < 0.05), these results are the first findings on the reproductive physiology of the shortfin silverside fish, suggesting an early steroidogenic activity in immature females. Furthermore, the species maintains its hormonal capacity during the post-spawning season, as it is supported by their follicular composition. The aforementioned will allow to have a better understanding of the mechanisms involved in their reproductive processes and to improve the mechanisms utilized to control their reproduction and for the production of eggs and larvae in culture conditions.

Se analizó el primer ciclo reproductivo de 50 hembras de pez blanco, Chirostoma humboldtianum (Valenciennes), en condiciones de cultivo. Se describieron los estadios de desarrollo ovárico y los índices gonadosomático (IG) y hepatosomático (IH). Se realizó una descripción histológica de los ovarios y se midieron los niveles séricos del estradiol (E2) y de la 17α-hidroxi -4-pregnen-3-ona (17-P4) por radioinmunoanálisis. Los resultados mostraron una primera época reproductiva de más de seis meses. Las hembras iniciaron sus desoves a la edad de un año. Se identificaron cuatro estadios de maduración ovárica (estadios I al IV) en la época de desoves y uno más (V) en la época no reproductiva, este último no tuvo un patrón de desarrollo definido. Los valores de los índices IG e IH mostraron una relación lineal positiva con el peso corporal únicamente en la época reproductiva (r² = 0.74 y r² = 0.86, P < 0.05, respectivamente). El análisis histológico de los ovarios indicó que la especie se clasifica como sincrónica por grupos múltiples. Los primeros estadios de maduración gonadal fueron caracterizados por ovarios cuyas poblaciones foliculares estuvieron compuestas predominantemente por folículos previtelinos. Conforme avanzó el proceso de maduración, se observaron todos los estadios de desarrollo folicular, con tendencia al incremento de folículos vitelinos y maduros. Durante el estadio I, las concentraciones de las hormonas esteroides sexuales circulantes de estradiol (E2) y de la 17α-hidroxi-4-pregnen-3-ona (17-P4) fueron altas (2.5 ± 0.7 ng/mL de E2 y 2.4 ± 0.3 ng/mL de 17-P4), se observó un decremento durante el estadio II y los valores más altos se registraron en el estadio IV (7.6± 2.1 ng/mL de E2 y 1.8 ± 0.9 ng/mL de 17-P4). Este mismo patrón fue observado durante la época de no desoves (ANDEVA P < 0.05); estos resultados son los primeros hallazgos en la fisiología reproductiva del pez blanco, los cuales sugieren una temprana actividad esteroidogénica en hembras inmaduras, además de que esta especie conserva su capacidad hormonal en la época posdesove, sustentada en su composición folicular. Lo anterior ayudará para comprender mejor los mecanismos involucrados en los procesos reproductivos y servirá de base para lograr su reproducción controlada y la obtención de huevos y larvas en condiciones de cultivo.

Histological ultrastructural aspects fat body virgin physogastric queens Melipona quadrifasciata anthidioides lepeletier, 1836 (HYMENOPTERA, APIDAE, MELIPONINI)

Variations in the morphology and biochemical content of insect fat body have been associated withmetabolic activity and the reproductive cycle (synthesis of vitellogenin). In social insects such as bees,the functional traits of fat body also differ between workers and queens. In this work, we used light andtransmission electron microscopy to examine the morphological features of fat body trophocytes of virginand physogastric mated queens of the stingless bee Melipona quadriafasciata anthidioides before and duringvitellogenesis. Virgin queens had few, small fat body cells in which lipid deposits predominate, and showedno evidence of biosynthetic activity or the uptake of exogenous substances. In contrast, the fat body cells ofphysogastric queens were almost completely devoid of lipids, exhibit a well-developed rough endoplasmicreticulum with an obvious intraluminal product, and contained Golgi stacks that release numerous vesicles.These ultrastructural findings were suggestive of proteosynthesis. However, there was no evidence for theaccumulation of synthesized material in the form of secretory granules. We conclude that the trophocytes ofvirgin and physogastric queens differ basically in their switch from a storage role in the former to a syntheticrole in the latter. In addition, the high level of vitellogenesis seen in egg-laying queens suggests that themain material synthesized is vitellogenin.

Identification of the stages of ovarian maturation of the Argentine hake Merluccius hubbsi Marini, 1933 (Teleostei: Merlucciidae): advantages and disadvantages of the use of the macroscopic and microscopic scales

The Argentine hake Merluccius hubbsi is a demersal-pelagic species on which few studies have been undertaken, despite its importance for the fisheries of the South-Southeastern Brazilian region, . The species is the most important commercial fishery resource in Uruguay and Argentina, where several studies have permitted the proper monitoring of the species. The ovarian maturation of the Argentine hake is analysed in this study. A scale of maturation is presented in the light of the oocyte development and the use of macro and microscopic scales of ovarian maturation are compared. It was detected that the oocyte types and derived structures present in the ovaries are similar to those already described both for the species and for teleosts in general; group synchronous development and multiple spawning are typical. The identification of ovarian maturation based only on external morphological characteristics of the gonads presents many errors, mainly in the characterization of the stages of maturation, which makes imperative the parallel analysis of the oocyte development, undertaken through histological ovarian cross-sections.

A merluza Merluccius hubbsi é uma espécie demerso-pelágica sobre a qual, apesar de sua importância nas pescarias da Região Sudeste-Sul do Brasil, poucos estudos foram realizados. Neste aspecto, destacam-se o Uruguai e a Argentina, países onde ela é o principal recurso pesqueiro e onde a existência de inúmeros estudos tem permitido o monitoramento da espécie. Neste estudo a maturação ovariana da merluza é analisada. Através do desenvolvimento ovocitário, apresenta-se uma escala de maturação e compara-se o emprego de escalas macro e microscópicas de maturação dos ovários. Constatou-se que os tipos de ovócitos e estruturas derivadas presentes nos ovários são semelhantes aos já descritos para a espécie e para teleósteos; o desenvolvimento dos ovócitos é sincrônico em grupos e a desova é parcelada. A identificação da maturação ovariana com base apenas em características morfológicas externas das gônadas apresenta muitos erros, principalmente na caracterização do estádio em maturação, sendo imprescindível a análise paralela do desenvolvimento ovocitário, realizada através de cortes histológicos dos ovários.