Previtellogenic Oocytes Research Articles

Mating behaviour and cholesterol nutritional strategies promoted ovarian development of female swimming crab (Portunus trituberculatus).

Female crabs enter a stage of rapid ovarian development after mating, and cholesterol is a substrate for steroid hormone synthesis. Therefore, in this experiment, an 8-week feeding trial was conducted to investigate the effects of mating treatments (mated crab and unmated crab) and three dietary cholesterol levels (0·09 %, 0·79 % and 1·40 %) on ovarian development, cholesterol metabolism and steroid hormones metabolism of adult female swimming crab (Portunus trituberculatus). The results indicated that crabs fed the diet with 0·79 % cholesterol significantly increased gonadosomatic index (GSI) and vitellogenin (VTG) content than other treatments in the same mating status. Moreover, mated crabs had markedly increased GSI and VTG content in the ovary and hepatopancreas than unmated crabs. The histological observation found that exogenous vitellogenic oocytes appeared in the mated crabs, while previtellogenic oocytes and endogenous vitellogenic oocytes were the primary oocytes in unmated crabs. The transmission electron microscopy analysis showed that when fed diet with 0·79 % cholesterol, the unmated crabs contained more rough endoplasmic reticulum and mated crabs had higher yolk content than other treatments. Furthermore, mating treatment and dietary 0·79 % cholesterol level both promoted cholesterol deposition by up-regulation of the mRNA and protein expression levels of class B scavenger receptors 1 (Srb1), while stimulating the secretion of steroid hormones by up-regulation of the mRNA and protein expression of steroidogenic acute regulatory protein (Star). Overall, the present results indicated that mating behaviour plays a leading role in promoting ovarian development, and dietary 0·79 % cholesterol level can further promote ovarian development after mating.

Long-term tributyltin exposure alters behavior, oocyte maturation, and histomorphology of the ovary due to oxidative stress in adult zebrafish

Tributyltin (TBT), an organotin endocrine-disrupting substance, is recognized as one of the important toxic environmental pollutants. The present study was carried out to investigate the toxic effects of TBT on behavior and the ovary of adult zebrafish with a focus on oxidative stress markers and oocyte maturation. Adult zebrafish were exposed to three different concentrations (125, 250, and 500 ng/L of water) of TBT for 28 days. TBT exposure produced a concentration-dependent negative effect on the body weight and behavior (anxiety-like symptoms) of adult zebrafish. Alterations in the activity of superoxide dismutase (SOD) and catalase (CAT), the total antioxidant capacity of ovarian tissue by the highest exposure level of TBT resulted in lipid peroxidation as indicated by increased malondialdehyde (MDA) level. The numbers of early-vitellogenic oocytes were significantly increased in zebrafish exposed to TBT as low as 125 ng/L. However, the numbers and size of fully-grown (mature) oocytes were significantly reduced in the highest exposure group only. Correlation between the MDA level and pre-vitellogenic oocytes in the 500 ng/L group indicated that lipid peroxidation prevented the maturation of pre-vitellogenic oocytes. TBT exposure produced significant histological changes in the ovary as evidenced by disturbed maturation of oocytes. In conclusion, TBT adversely affected the maturation of oocytes in zebrafish ovary through oxidative stress-mediated mechanisms.

Potential Implications of Acid-Sensing Ion Channels ASIC2 and ASIC4 in Gonadal Differentiation of Dicentrarchus labrax Subjected to Water Temperature Increase during Gonadal Development.

In this study, the expression and implication of acid-sensing ion channels 2 and 4 (ASIC2 and ASIC4) in the gonadal sex differentiation of Dicentrarchus labrax (D. labrax), subjected to increasing water temperatures during gonadal development, were evaluated. Two groups were selected: a control group (CG), in which the average water temperature was maintained at 15 °C and increased to 20 °C in 20 days until weaning; and an experimental group (EG), in which the water temperature was retained at 15 °C for 60 days; thereafter, the temperature was increased daily by 0.5 °C until it reached 20 °C up to the weaning time. Ten fish from the CG and 13 fish from the EG were sampled randomly on the 335th day after hatching (dph). A higher percentage of gonad differentiation in ovaries rather than in testes was observed in the EG compared to the CG (p = 0.01). ASIC2 and ASIC4 were detected for the first time in D. labrax ovaries by indirect immunofluorescence. Both ASIC2 and ASIC4 were expressed in previtellogenic oocytes of ovaries and in scattered cells within some testes, and were most likely intratesticular previtellogenic oocytes in both the CG and EG groups. The CG group showed a higher expression of ASIC4 than the EG cohort (p < 0.05). The results gathered in this study revealed the capacity of water temperature to influence both gonadal differentiation and growth in this gonochoristic fish species, and suggests the possible role of ASIC2 and ASIC4 in gonad differentiation and gamete development in D. labrax.

Trade-off between growth and reproduction in Argopecten purpuratus (L.) scallops exposed to medium-term hypoxia and acidification

Peruvian bays that are home to aquatic organisms of commercial interest are increasingly affected by hypoxia and low pH events. These stressors produce unfavorable conditions for the aquatic fauna, leading to mass mortality events. In this study, we evaluated the ecophysiological responses of the scallop Argopecten purpuratus exposed to moderate hypoxia (20% DO saturation) and low pH (OA, pH 7.4) using a 2 × 2 factorial design. We found that a 51-day exposure to low pH (OA treatment) had greater negative effects on A. purpuratus performances than hypoxia (lower survival and decreased clearance and calcification rates), but stimulated early gonad maturation. The survival rate was 1.3-fold higher under hypoxia than under acidic treatment. The interaction between hypoxia and low pH had an antagonistic effect on survival, since the combined treatment (HOA) resulted in lower scallop mortality than the single stress treatments. Calcification was negatively affected by all stress treatments, whereas hypoxia and OA both stimulated gametogenesis. OA treatment resulted in higher frequencies of previtellogenic and vitellogenic oocytes, greater gonad coverage area, and lower frequency of atretic oocytes, suggesting higher reproductive potential. HOA was positively related to oocyte development and high frequency of post-vitellogenic and atretic oocytes. These results suggest that, due to hypoxia and low pH, feeding is reduced and energy allocation prioritizes scallop gonad maturation. This trend would have negative effects on scallop growth and calcification, while increased reproduction under environmental stress could mitigate the effect on recruitment.

Characterization of Two Gonadal Genes, zar1 and wt1b, in Hermaphroditic Fish Asian Seabass (Lates calcarifer)

Zygote arrest-1 (Zar1) and Wilms’ tumor 1 (Wt1) play an important role in oogenesis, with the latter also involved in testicular development and gender differentiation. Here, Lczar1 and Lcwt1b were identified in Asian seabass (Lates calcarifer), a hermaphrodite fish, as the valuable model for studying sex differentiation. The cloned cDNA fragments of Lczar1 were 1192 bp, encoding 336 amino acids, and contained a zinc-binding domain, while those of Lcwt1b cDNA were 1521 bp, encoding a peptide of 423 amino acids with a Zn finger domain belonging to Wt1b family. RT-qPCR analysis showed that Lczar1 mRNA was exclusively expressed in the ovary, while Lcwt1b mRNA was majorly expressed in the gonads in a higher amount in the testis than in the ovary. In situ hybridization results showed that Lczar1 mRNA was mainly concentrated in oogonia and oocytes at early stages in the ovary, but were undetectable in the testis. Lcwt1b mRNA was localized not only in gonadal somatic cells (the testis and ovary), but also in female and male germ cells in the early developmental stages, such as those of previtellogenic oocytes, spermatogonia, spermatocytes and spermatids. These results indicated that Lczar1 and Lcwt1b possibly play roles in gonadal development. Therefore, the findings of this study will provide a basis for clarifying the mechanism of Lczar1 and Lcwt1b in regulating germ cell development and the sex reversal of Asian seabass and even other hermaphroditic species.

The effects of estradiol‐17β on the sex reversal, survival, and growth of Red Shiner and its use in the development of YY individuals

AbstractObjectiveThe Red Shiner Cyprinella lutrensis is one of the most prolific and ecologically destructive invasive fish species in the southwestern United States. The production and release of YY individuals as Trojan sex chromosome carriers can theoretically eradicate invasive fish populations by eventually eliminating phenotypic females.MethodsThe YY individuals are typically produced through hormonally induced sex reversals and selective breeding of subsequently feminized males. We tested three dosages of estradiol‐17β (E2)‐treated diets (50, 100, and 150 mg of E2 per kg of diet) administered to sexually immature Red Shiner for various durations to determine their effectiveness at feminizing Red Shiner cohorts. Survival, growth, and gonadal development were assessed for each treatment.ResultAll E2 treatments had minimal, if any, detrimental effects on the growth and gonadal development of Red Shiner. The 50‐mg dosage lasting from 2 to 120 days posthatch achieved a 100% feminization rate while using the lowest amount of E2; therefore, this dosage and treatment interval are recommended when attempting Red Shiner feminization under these rearing conditions. Feminization of males allowed for the spawning of neofemales (FXY) with wild‐type males (MXY), which resulted in the first putative YY Red Shiner. The YY verification crosses (n = 20) resulted in predominately male offspring (189 males/191 offspring) except for (1) an intersex individual from an MYY × FXX cross with two previtellogenic oocytes in its testis and (2) a single female that may have resulted from an inbred cross between an XY male and a YY female or from an unknown autosomal or environmental effect on sexual phenotype.ConclusionMore progeny tests with inbred and outbred crosses should be conducted to determine the prevalence of female offspring from YY individuals and how this may impact an eradication strategy featuring releases of YY Red Shiner.

Alteration in the histology of gonads and hormone levels of fish Channa punctatus on exposure to 17-α ethynylestradiol

It has been demonstrated that effluents discharged from sewage treatment facilities allow synthetic estrogens, widely employed in contraceptives and other pharmaceutical applications, to reach the aquatic environment. One of the most biologically active xenoestrogens in the aquatic environment is the synthetic hormone 17-α ethynylestradiol(EE2), raisingreproductive issues in the fish population. The present study chose Channa punctatus fish to study the effects of EE2 on gonad histology and the calculation of estrogen and testosterone levels. The experimental setting was separated into four groups, with Group I as control with 0 ng/L of EE2. The other three groups viz., Group II to IV, had 5 ng/L, 10 ng/L, and 20 ng/L concentrations of EE2, respectively, and samples were obtained after the durations of 7, 14, 21, and 28 days. The findings showed that as 17-α ethynylestradiol concentrations increased, testosterone levels decreased from 10.88±0.24 ng/dL to 4.91±0.24 ng/dL,while estrogen levels increased from 42.7±2.22 pg/mL to 120.18±4.54 pg/mL. The number percentage of primary growth oocytes, previtellogenic oocytes and vitellogenic oocytes in the histology of ovary decreased from 28.45±1.42 to 10.43±0.47; 16.22±0.81 to 3.15±0.16 and 14.81±0.89 to 2.14±0.11 respectively while in testis, the number percentage of spermatogonia, spermatocytes decreased from 21.4±0.86 to 15.1±0.68 and 97.1±3.88 to 54.2±3.25. In contrast, the percentage of mature spermatids increased from 20.8±0.94 to 40.2±2.41. Since synthetic estrogens adversely affect aquatic animals, especially fish, they should be treated properly before releasing them into the aquatic environment.

Production of neofemale by 17β-estradiol and YY super-male breeding in mandarin fish (Siniperca chuatsi)

It is well known that mono-sex culture has the advantages of a more rapid growth rate, more uniform size and lower energy consumption of gonadal development in fish. In production practice, male mandarin fish (Siniperca chuatsi) show great aquaculture value after the overwintering stage. As a species with an XY-type sex determination system, feminizing genetically male fish into physiological female fish and breeding YY supermales is a common method used to produce all-male populations. In this study, we systematically investigated feminization of mandarin fish by indirect feeding of 17β-estradiol (E2) at different doses (200, 400 and 600 mg/kg). Neo-females were successfully yielded in all E2-treated concentrations. The sex-reversed ratios of the 200, 400 and 600 mg/kg E2-treated groups were 40%, 87% and 100%, respectively. From 45 to 135 dat (days after treatment), the gonads in the 600 mg/kg E2-treated group consisted of oogonium and previtellogenic oocytes. At 165 dat, early vitellogenic oocytes were observed in the gonads of the 600 mg/kg E2-treated group. Regarding the expression of sex-related genes, 600 mg/kg E2-treated mandarin fish presented a different gene expression pattern from that of normal males. Moreover, the growth and development status were also analysed in XY normal males and YY supermales at 360 dph, and YY supermales showed a higher number of lumina and significantly lower growth performance than XY normal males. In conclusion, we have established a technique for efficiently inducing male to female sex reversion and YY supermale breeding of mandarin fish, which provides a great foundation for the production of all-male mandarin fish.

The Effect of Different Photoperiods on The Oogenesis of Fighting Fish (Betta Splendens)

The demand for fighting fish Betta splendens continues to increase every year, this encourages farmers to develop technology in the production process. Accelerating reproduction is one solution to support production demand. One technology prior to this is photoperiod. The aim of this research was to evaluate different photoperiod treatments on the stages of betta fish ovarian development. The method used is completely randomized design (CRD) to test 3 different treatments in quintuplicates. Treatment P1 (10-h Light L :14-h Dark D), P2 (14-h L :10-h Dark D), and P3 (Control, ambient lights). Observation of the oogenesis stages of betta fish was carried out through histological preparations classic and calculating the proportions of oogenesis stages. Data were then analyzed statistically (ANOVA) with 0.05 level. The research results showed that treatment P2 was significantly different from P1 and P3, due to domination of previtellogenic oocytes (22.05%), oocyte exogenous vitellogenin (22.89%), and hydrated vitellogenin oocytes (25.24%). Then followed by treatment P1 (10L; 14D) presented previtellogenic oocytes ​​(20.86%), exogenous vitellogenin oocyte (20.17%), and hydrated vitellogenin oocytes (21.82%). In Control, ovaria contained previtellogenic oocytes (19.43%), exogenous vitellogenin oocytes (18.79%), and hydrated vitellogenin oocytes (19.21%). This indicated that under such photoperiod treatments fish kept on adept to develop their ovaria. Moreover, fish matured their ovaria and were apt to spawn.

Effects of CRISPR/Cas9-mediated dnd1 knockout impairs gonadal development in striped catfish

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology allows for the generation of loss-of-function mutations to enable efficient gene targeting to produce desired phenotypes, such as the production of germ cell-free fish. This technology could provide several applications for aquaculture and conservation of fisheries resources, such as the prevention of overpopulation in fish culture and gene flow from escaped farmed fish into wild populations and the production of germ cell-free recipient larvae for germ cell transplantation. This study aimed to develop CRISPR/Cas9 mediated dead-end 1 (dnd1) knockout techniques for striped catfish (Pangasianodon hypophthalmus). To optimise CRISPR/Cas9-induced dnd1 knockout, three single-guide RNAs (sgRNAs) were designed to target upstream sequences of start codon of the dnd1 gene. A combination of two concentrations of each sgRNA (100 and 200 ng/µl) and three concentrations of Cas9 (100, 250, and 500 ng/µl) was microinjected into fertilised striped catfish eggs. These sgRNAs/Cas9 could induce indel mutations and lower the primordial germ cell (PGC) numbers. Histological analyses indicated that sgRNA3 targeting upstream and nearest to the start codon at 200 ng/µL and Cas9 at 500 ng/µL showed the lowest PGC number. The reduction in PGC number was confirmed by in situ hybridisation using antisense dnd1 and vasa probes. All sgRNA/Cas9 combinations reduced the expression of dnd1, cxcr4b, dazl, nanos1, nanos2, and vasa, and the lowest expression levels were observed in gonads obtained from fish injected with 200 ng/µL sgRNA3 and 500 ng/µL Cas9 (P < 0.05). In addition, at 1 year of age, a significantly lower gonadosomatic index was observed in fish injected with all sgRNA and Cas9 at 500 ng/µL. Moreover, compared to the control fish, the ovaries and testes presented different morphologies in the sgRNA/Cas9-injected fish, that is, few previtellogenic oocytes in the ovary and spermatogonial cell-less testes. In conclusion, CRISPR/Cas 9 targeting dnd1 knockout at the upstream sequences of start codon was achieved, which resulted in the downregulation of dnd1 and lowered PGC number.

Molecular and functional characterization of ribosome protein S24 in ovarian development of Macrobrachium nipponense

Ribosomal proteins (RPs) have mang extraribosomal functions including regulation of ovarian development in some organisms. In order to solve the problem of rapid ovarian maturation in Macrobrachium nipponense aquaculture, this study identified a RPS24 (MnRPS24) gene from M. nipponense, which encodes a protein of ββαβαααα folding structure type. MnRPS24 exhibited the greatest expressions in the female adult stage among the six growth stages, in the ovary among the nine tissues, and in the stage I ovary among the six ovarian development stages. The MnRPS24 protein located in the cytoplasm of oogonia, previtellogenic and early-vitellogenic oocytes, and the follicular cells surrounding the oocytes. The expression of the vitellogenin (MnVg), vitellogenin receptor (MnVgr), cell cycle protein B (MnCyclin B) and cell division cyclin 2 (MnCdc2) genes were increased by recombinant MnRPS24 protein incubation. Conversely, the expression of the Wee1 kinase (MnWee1) gene was decreased. MnRPS24 gene silencing downregulated the expression for MnVg, MnVgr, MnCyclin B and MnCdc2 and upregulated the expression for MnWee1. Furthermore, MnRPS24 gene silencing delayed the vitellogenesis of oocytes, halting the progression of ovarian development. The findings of this research demonstrate that MnRPS24 could potentially function as a stimulator in promoting the development of ovaries in M. nipponense.

Oocyte development, packing in the ovary, and spawning pattern of squid Lolliguncula panamensis in the Mexican South Pacific

Squid Lolliguncula panamensis Berry, 1911 is of commercial and ecological importance in the Gulf of Tehuantepec (southeastern Mexico). This study examines the mode of oocyte development, packing in the ovary, and spawning pattern of this understudied species. Lolliguncula panamensis presents a synchronous oocyte growth in batches, with multiple spawnings, with the presence of post-ovulatory follicles and atretic follicles, including the constant presence of pre-vitellogenic and vitellogenic oocytes. The size frequencies of oocytes of ripe ovaries exhibit a pronounced morphological and size separation between the late yolk stage and mature oocytes, suggesting that the oocytes pass through this size range very fast. These oocytes adopt a geometrical shape that makes their packing efficient for spawning. One three-dimensional packing of vitellogenic oocytes (scutoid type) in the ovary was observed. The results of our study suggest that the spawning behavior of Lolliguncula panamensis may constitute an opportunistic reproductive strategy in which individuals present multiple spawnings over a brief period. We suggest that this strategy might be a response to complex regional oceanographic conditions of the Gulf of Tehuantepec.

Ovary of Zebrafish during Spawning Season: Ultrastructure and Immunohistochemical Profiles of Sox9 and Myostatin.

This study sought to examine the ovarian cellular and stromal components of the zebrafish (Danio rerio) throughout the spawning season using light and electron microscopic tools. The ovaries of zebrafish showed oocytes in all stages of follicular development and degeneration (atresia). Six stages of oogenesis were demonstrated: oogonia, early oocytes, late oocytes, vacuolated follicles, the yolk globule stage (vitellogenesis), and mature follicles. The SOX9 protein was expressed in the ooplasm of the primary and previtellogenic oocytes and the theca cell layer of the mature follicles. Myostatin was expressed in the granulosa and theca cells. Many stem cells in the ovarian stroma expressed myostatin and SOX9. During the spawning season, the EM results indicated that the zona radiata increased in thickness and was crossed perpendicularly by pore canals that contained processes from both oocytes and zona granulosa. The granulosa cells contained many mitochondria, rER, sER, and vesicles. Meanwhile, the thecal layer consisted of fibroblast-like cells. Atretic follicles could be demonstrated that involved both oocytes and their follicular walls. Several types of cells were distinguished in the ovarian stroma, including mast cells, telocytes, lymphocytes, fibroblasts, endocrine cells, macrophages, adipocytes, dendritic cells, and steroidogenic (stromal) cells. The ovary of the zebrafish serves as a model to investigate follicular development.

Expression pattern of AGPATs isoforms indicate different functions during the triacylglyceride synthesis in Chinese mitten crab, Eriocheir sinensis

The 1-acylglycerol-3-phosphate acyltransferase (AGPAT) acts as a crucial enzyme in the process of triacylglycerol (TAG) synthesis, enabling the acylation of lysophosphatidic acid (LPA) into phosphatidic acid (PA). In order to decode the distinctive roles of AGPAT isoforms in the TAG production pathway, three AGPAT isoforms were detected for the first time in the Chinese mitten crab Eriocheir sinensis (Es-agpat2, Es-agpat3, and Es-agpat4). The mRNA levels of Es-agpat2 and Es-agpat4 demonstrated a conspicuous presence in the hepatopancreas, with subsequent high levels in the heart, muscle, and thoracic ganglion. On the other hand, the thoracic ganglion exhibited abundant levels of Es-agpat3, while other tissues recorded relatively low expression levels. Observing the molting cycle of E. sinensis, the hepatopancreas showed minimum expression levels of Es-agpat2 and Es-agpat4 at stage A/B. A peak at stage C was noted, which was then followed by a gradual drop until stage E. For the ovarian development cycle, stage II witnessed the maximum expression level of Es-agpat2 and Es-agpat4, succeeded by a sharp fall in stage III. After this, there was an increasing trend from stage III up to stage V. Expression of Es-agpat3 in the hepatopancreas was consistently lower than Es-agpat2 and Es-agpat4 during either the molting or ovarian development. However, in terms of ovarian expression, Es-agpat3 outperformed Es-agpat2 and Es-agpat4. It exhibited a steep increase in expression, peaking at stage II and subsequently diminishing. In situ hybridization (ISH) revealed that in stages II and IV hepatopancreas, Es-agpat4-mRNA was primarily located in fibrillar cells (F cell) and resorptive cells (R cell), with no signal from Es-agpat3. During stage II of ovarian development, both Es-agpat3-mRNA and Es-agpat4-mRNA were located in the cytoplasm of previtellogenic oocyte (PRO) and endogenous vitellogenic oocyte (EN), with no expression at stage IV. Additionally, the silencing of Es-agpat2 and Es-agpat4 caused a downward trend in the expression levels of all subsequent genes in the E. sinensis TAG synthesis pathway. To sum up, these findings suggest that the three Es-agpats may have unique functions in TAG synthesis during either the molting process or ovarian maturation of E. sinensis.

Asymmetry in previtellogenic and early vitellogenic oocytes, ultrastructure of follicular cells and egg envelope in the pigmented sterlet, Acipenser ruthenus L. 1758 (Chondrostei, Acipenseriformes).

Ovarian follicles of sterlets (Acipenser ruthenus) are composed of a single oocyte surrounded by follicular cells (FCs), basal lamina, and thecal cells. Previtellogenic oocytes are polarized. Homogeneous ooplasm (contains ribosomes) and granular ooplasm (comprises nuage aggregations of nuclear origin, rough endoplasmic reticulum (RER), Golgi complexes, ribosomes, and mitochondria) are distinguished. Granular ooplasm is initially located near the nucleus, contacts the plasma membrane of the oocyte (oolemma) and forms a thin layer underneath its entire perimeter. Next, a ring that surrounds the nucleus is formed and sends strands directed toward the oolemma. The lipid body composed of lipid droplets forms adjacent to this ring. Later, the granular ooplasm and strands enlarge toward the oolemma, lipid body disperses, and homogeneous ooplasm is no longer present. A thin cortical ooplasm is formed underneath the oolemma and does not contain any organelles. The oocyte nucleus moves to the center. The nucleoplasm contains lampbrush chromosomes, nuclear bodies, and multiple nucleoli. Early vitellogenic oocytes are polarized, too. Three regions in the ooplasm are distinguished: the perinuclear (contains lipid droplets near the nuclear envelope), the endoplasm (contains yolk platelets and lipid droplets), and the periplasm (contains yolk spheres, pigment granules, and microtubules). In all these regions the RER, Golgi complexes, nuage, and mitochondria are present. Micropinocytotic vesicles, Golgi vesicles and precursors of the internal layer of the egg envelope are in the cortical ooplasm. Some FCs delaminate from the follicular epithelium, degenerate and vesicles are released into the perioocytic space. They may contain precursors of egg envelope and may be involved in "cell-cell" communication. The egg envelope (zona radiata, zona pellucida) is made up of three layers: the vitelline envelope (inner layer), the middle layer, and the outer layer. In its deposition, both the oocyte and FCs are engaged.

Assessing the ovarian development of Macrobrachium amazonicum (Heller, 1862) phenotypes by means of an integrative analysis

Macrobrachium amazonicum is a species of economic interest with a wide distribution in the Americas and high morphological and reproductive variability. Three phenotypes can be observed in this species: i) large-size amphidromous, ii) large-size and iii) small-size hololimnetic prawns. In the present work, the morphological, histochemical and ultrastructural aspects of ovarian development in the three phenotypes were comparatively analyzed. In addition, the interaction between the ovary and the hepatopancreas was investigated in these phenotypes through the use of gonadosomatic (GSI) and hepatosomatic (HSI) indices. Despite the morphological differences and different reproductive strategies adopted by the females, the macroscopic, histochemical and ultrastructural patterns of ovarian development showed no differences between the phenotypes. The ovaries were macroscopically classified into five stages of development (I to V). In early stages (I and II), the ovaries are full of oogonia, previtellogenic oocytes and oocytes in primary or endogenous vitellogenesis. At these stages, the rough endoplasmic reticulum (RER) produces a granular electron-dense material and sends it to the Golgi apparatus, where it will be modified, compacted and transformed into immature yolk granules. From stage III, secondary or exogenous vitellogenesis begins (with no interruption of endogenous vitellogenesis), where follicular cells nourish the oocytes and extracellular material is absorbed by endocytic vesicles, which fuse with immature yolk granules (forming mature granules) or with existing mature yolk granules. In stages IV and V, secondary vitellogenesis continues and mature yolk granules progressively occupy the cytoplasm. In M. amazonicum, the patterns of increase in oocyte diameter are quite similar between phenotypes, being greater in the small-size phenotype. This is related to the formation of larger oocytes/eggs and the production of large lipid reserves for their larvae. Changes in GSI and HSI during ovarian development show strong similarity between phenotypes, supporting the results obtained by histology and ultrastructure. Females in stages III and IV mobilize hepatopancreas reserves for ovarian maturation, which justifies the higher HSI values recorded in these stages. On the other hand, females in stage V show higher GSI and lower HSI values, indicating a mobilization of resources for the end of ovarian development as the females are ready to spawn.

Molecular Markers of Ovarian Germ Cells of Banana Prawn (Fenneropenaeus merguiensis).

The banana prawn (Fenneropenaeus merguiensis) is a valuable prawn in the worldwide market. However, cultivation of this species is limited owing to the difficulty in culture management and limited knowledge of reproduction. Therefore, we studied the gene expression and molecular mechanisms involved in oogenesis for elucidating ovarian germ cell development in banana prawns. The tissue-specific distribution of certain genes identified from previous transcriptome data showed that FmCyclinB, FmNanos, and nuclear autoantigenic sperm protein (FmNASP) were only expressed in gonads. The in situ hybridization (ISH) of these three genes showed different expression patterns throughout oogenesis. FmCyclinB was highly expressed in pre-vitellogenic oocytes. FmNanos was expressed at almost the same level during oogenesis but showed the most expression in late pre-vitellogenic stages. Based on the highest expression of FmCyclinB and FmNanos in mid pre-vitellogenic and late pre-vitellogenic oocytes, respectively, we suggested that FmNanos may suppress FmCyclinB expression before initiation of vitellogenesis. Meanwhile, FmNASP expression was detected only in pre-vitellogenesis. Moreover, quantitative real-time polymerase chain reaction (qRT-PCR) analysis of FmNASP expression was supported by FmNASP ISH analysis based on high expression of FmNASP in sub-adult ovaries, which contain most of pre-vitellogenic oocytes. In this study, we found three reliable ovarian markers for banana prawns and also found a dynamic change of molecular mechanism during the sub-stage of pre-vitellogenesis. We determined the expression levels of these genes involved in oogenesis. Our findings provide information for further studies on banana prawn reproduction which may assist in their cultivation.

Pubertal sexual development and endpoints for disrupted spermatogenesis in the model Xenopus tropicalis

Peripubertal models to determine effects of anti-androgenic endocrine disrupting chemicals are needed. Using the toxicological model species Xenopus tropicalis, the aims of the study were to 1) provide data on sexual maturation and 2) characterise effects of short-term exposure to an anti-androgenic model substance. Juvenile (2.5 weeks post metamorphosis old) X. tropicalis were exposed to 0, 250, 500 or 1000 µg flutamide/L (nominal) for 2.5 weeks. Upon exposure termination, histology of gonads and Müllerian ducts was characterised in detail. New sperm stages were identified: pale and dark spermatogonial stem cells (SSCs). The testes of control males contained spermatozoa, indicating pubertal onset. The ovaries were immature, and composed of non-follicular and pre-vitellogenic follicular oocytes. The Müllerian ducts were more mature in females than males indicating development/regression in the females and males, respectively. In the 500 µg/L group, the number of dark SSCs per testis area was decreased and the number of secondary spermatogonia was increased. No treatment effects on ovaries or Müllerian ducts were detected. To conclude, our present data provide new knowledge on spermatogenesis, and pubertal onset in X. tropicalis. New endpoints for evaluating spermatogenesis are suggested to be added to existing assays used in endocrine and reproductive toxicology.

β-Carotene Analysis and Histological Investigation on Different Egg Stages of Anomuran Crab, Emerita Asiatica (H. Milne Edwards, 1837)

Histological investigation and β-carotene analysis of different egg stages of anomuran crab, Emerita asiatica was studied. This investigation characterized the morphological and histological features of the ovaries; eggs and also resulted in the identification of the following structures such as Previtellogenic Oocyte, Vitellogenic Oocytes, Atretic Oocyte, matured Oocyte and its associated structures which are quiet common structures that are found in almost every Sand Crab. This study can be further used in studying the reproductive biology of this Sand Crab (Emerita asiatica). It can also be used in determining the structural and morphological changes that can happen in gonads due to the action of various microorganisms such as bacteria, viruses and fungi by artificial infections. The observation described in this study will serve as a reference for future research aimed at studies based on environmental factors, temperature, climatic conditions, and feeding that might have an effect on this species. The conversion of β-carotene into vitamin A (retinol), for clear eyesight and healthy eyes, a robust immune system, and healthy skin and mucous membranes, we require vitamin A. Large amounts of vitamin A can be hazardous, but our bodies only make as much of the vitamin from β-carotene as is required. The sand crab serves as a different source of β-carotene than vitamin A, even though E. asiatica is not widely used. Though E. asiatica is not a commercially viable crab, it plays a vital role in the environment to maintain a stable marine ecosystem. Several steps and preventive measures should be taken to conserve these members of the marine food chain to have a stable ecosystem and also to protect this species from extinction. Further, this study can potentially benefit as baseline information for the future research on the species.

Reproductive Biology of the Golden Cuttlefish Sepia esculenta (Cephalopoda, Sepiida)

The golden cuttlefish Sepia esculenta is the one of most abundant cuttlefish species around south-east Asia and has a high commercial value. Despite its wide distribution and high commercial value, its reproductive biology is still poorly understood. This study was based on 25 males and 6 females. The potential fecundity (PF) of females was 1701–3719 oocytes, which was an increase, as compared to the previously known values. The oocyte resorption reached up to 13.2% of fecundity. The ovulation pattern was group-synchronous, with a predominance of previtellogenic oocytes. The pre-meiotic and primary growth oocyte phases were absent in mature females. The number of spermatophores carried by an individual male was 146–1698 (length 9–20 mm). The spermatophores were characterised by a cement body consisting of conical oral and cylindrical aboral parts. The ontogenetic changes in the spermatophores and their parts were recorded for the first time in the order of Sepiida. Their sperm content and their adhesive abilities also increased during ontogenesis. The data obtained in the present study significantly increased and corrected the existing knowledge of S. esculenta biology. Moreover, these data help to explain the general patterns of reproductive biology in cuttlefish, as well as in Cephalopoda as a whole.