Stages Of Ovarian Development Research Articles

Pubertal moult and ovarian development pattern of the swimming crab, Portunus trituberculatus, using Single Crab Box Culture

The pubertal moult is the key step in the transition from a sub-adult to an adult crab (Xuan et al., 2014). In the swimming crab Portunus trituberculatus (Miers, 1876), it has been documented that successful mating can happen soon after the female has finished her pubertal moult, with male spermatophores being transferred into the female spermatheca (Wu et al., 2007). Then female crabs enter into the stage of rapid ovarian development, which is initiated only after the crab has successfully mated (Wu et al., 2007). Due to the strong market demand and high returns of potential profit for P. trituberculatus, its pond culture has expanded along the east coast of China (Che et al., 2018). However, cannibalism during the moulting period is high, leading to a reduced survival percentage and negatively affecting overall farming efficiency (Duan et al., 2011; Hou et al., 2016).

Comparative metabolomics analysis of ovarian developmental stages in Macrobrachium nipponense

Rapid sexual maturity of female Macrobrachium nipponense is a severe problem for the aquaculture industry. To date, there have been only transcriptome studies investigating ovarian development, and studies using other tools, such as metabolomics are lacking. Metabolomics reveals changes in the level of metabolites in tissues in relation to current physiological characteristics, and can yield valuable insight into the growth and development of organisms. In this study, we systematically analyzed 15 samples from five different ovarian developmental stages in M. nipponense to learn more about how metabolites change over reproduction. Gas chromatography/time-of-flight mass spectroscopy revealed an array of different compounds and 83–162 pathways depending on the stage. Furthermore, 89 metabolites and 14 pathways were significantly different across stages. It is hypothesized that N-acetyl-N-formyl-5-methoxykynurenamine, ascorbate, fructose-2,6-bisphosphate, cortexolone and other metabolites that significantly differed by stage are regulated by hormones and are closely related to ovarian development. However, for other metabolites that changed with development, such as cytidine and xanthine, an association with ovarian development has yet to be revealed. Quantitative polymerase chain reaction was used to correlate gene changes to metabolites in the pathway for biosynthesis of plant secondary metabolites. We found that the TCA cycle rate may be the cause of female miniaturization during the reproductive period, and that the control of fatty acid content via aquaculture nutrition may be an exogenous tool for regulatory control of maturation. This study provides a systematic and comprehensive metabolomics analysis of ovarian development in M. nipponense and lays a foundation for addressing the problem of rapid sexual maturity.

Timing of oocyte recruitment within the ovulatory cycle of Macedonian shad, Alosa macedonica, a batch spawning fish with indeterminate fecundity

The recruitment of primary growth (PG) oocytes to the secondary growth (SG) phase within the ovulatory cycle, i.e. time interval between two sequential ovulation/spawning events, has rarely been examined in detail in a quantitative manner for batch spawning fishes with indeterminate fecundity. In the present study we analyzed the ovarian dynamics of Macedonian shad, Alosa macedonica, an iteroparous batch spawning clupeid with indeterminate fecundity, with the main goal to define the timing of PG recruitment and relate it to the ovulatory cycle. The latter was classified into four different phases (pre-ovulatory, running, post-ovulatory, intermediate) through postovulatory follicles and presence/absence of hydrated oocytes. Various indices of ovarian dynamics, including the formation and mean diameter of the advanced oocyte batch, the ovarian developmental stage, the oocyte size modality and the gonadosomatic index, varied among the ovulatory phases, evidencing cyclicity. Relative fecundity of newly recruited SG oocytes was used as an index of PG recruitment intensity and was shown to follow a specific pattern within the ovulatory cycle; PG recruitment occurred in a stepwise manner and in parallel with ovulation of the advanced oocyte batch, and synchronization of these two processes kept the ovary in a state of dynamic equilibrium.

Induced spawning and embryonic and early larval development of bonefish (Albula vulpes).

Bonefish (Albula vulpes L.) are a highly prized sport fish. Despite their economic importance, populations in the Florida Keys and Caribbean are in decline, with the early life history undescribed. Injections of carp pituitary extract into A. vulpes during the advanced stages of ovarian development induced ovulation and spawning. Embryos were sampled hourly until hatching into undeveloped, yolk-sac leptocephalus larvae. These larvae survived 56 h post-hatch, when myomeres and eyes were developing but not the mouth. These results inform future research on the reproduction and early life history of A. vulpes.

MiR-431 regulates granulosa cell function through the IRS2/PI3K/AKT signaling pathway.

MicroRNAs (miRNAs) regulate the functions of granulosa cells by interacting with their target mRNAs. Insulin receptor substrate 2 (IRS2) is one of the targets of miR-431 and can be regulated by ovarian hormones. However, the role of miR-431 and the associated signal transduction pathway in ovarian development has not been studied previously. In this study, we first analyzed the expression of miR-431 and IRS2 following stimulation with pregnant mare serum gonadotropin (PMSG) during the estrous cycle or different stages of ovarian development in mice. Subsequently, we investigated the role, function, and signaling pathway of miR-431 in the human granulosa cell line, COV434. The results showed that follicle stimulating hormone (FSH) gradually decreased miR-431 levels, induced IRS2, and promoted pAKT expression. Moreover, miR-431 overexpression and IRS2 knockdown attenuated AKT activation, inhibited cell proliferation, and decreased estradiol (E2) and progesterone (P4) synthesis. Further, luciferase reporter assay demonstrated that IRS2 was a direct target of miR-431. In conclusion, this study demonstrated that miR-431 regulates granulosa cell function through the IRS2/PI3K/AKT signaling pathway.

TGF-β1 sustains germ cell cyst reservoir via restraining follicle formation in the chicken.

The transforming growth factor β (TGF-β) superfamily members are important molecules that regulate many ovarian functions under normal physiological and pathological conditions. TGF-β1 and its receptors are highly expressed in the ovarian cells of many species. However, the effect of TGF-β1 on the capacity of the avian germ cell reservoir remains unknown. In this study, 5-day-old chicks were injected with TGF-β1 (2.5, 12.5, and 62.5 μg/kg body weight) for 3 days to assess the effect of TGF-β1 on early follicle development. Morphological analysis showed that treatment with TGF-β1 (12.5 μg/kg) increased the number of germ cell cysts and reduced the number of primordial and growing follicles. The diameter and area of oocytes and follicles were decreased after TGF-β1 treatment. Immunohistochemical staining of the proliferating cell nuclear antigen revealed that the ratios of the positive somatic and granulosa cells were decreased by 16.2% and 2.48%, respectively. Furthermore, more apoptotic cells were observed in the TGF-β1 group than thoseof the control by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. In addition, we cultured the 5d chicken ovaries for 3 days in vitro and found that treatment with TGF-β1 (10 ng/mL) manifested similar results as the in vivo experiment. However, the negative effect of TGF-β1 on early ovary development was rescued by treatment with a TGF-βR1 inhibitor SD208, resulting in increased expression of steroidogenic enzymes and cell cycle-regulating proteins. In conclusion, TGF-β1 could maintain the germ cell reservoir by restraining follicle activation involving reduced cell proliferation and steroidogenic enzymes gene expression at the early stage of ovarian development.

Gonadosomatic Index, Oocyte Development and Fecundity of the Snakehead Fish (Channa striata) in Natural River of Mae La, Singburi Province, Thailand.

Reproductive cycle of snakehead fish (Channa striata) is necessary to inform management plans of the natural fish. This work aimed to study the gonadosomatic index (GSI), oocyte development and fecundity of C. striata in the Mae La River, Singburi province, Thailand from November, 2017-October, 2018. GSI was calculated from gonadal weight and body weight. Ovarian sections derived from standard histological methods were used to evaluate oocyte development, respectively. Fecundity was measured using the gravimetric method and the relationships of fecundity-total length, fecundity-body weight and fecundity-ovarian weight were evaluated. The highest average GSI values for females and males were found in July (6.15±0.20 and 0.14±0.12, respectively). Ovarian histology revealed 4 stages of ovarian development: (1) immature, (2) maturing, (3) mature and (4) spent. Stages 1, 2, 3 and 4 were observed for 11, 10, 6 and 2 months, respectively. The highest percentages of immature, mature and spent oocytes were found in October (93.33%), July (89.56%) and August (33.33%), respectively. Fecundity ranged from 4,160-46,890. Fecundity varied depending on total length, body weight and ovarian weight, with correlation coefficients (R) of 0.986, 0.960 and 0.989, respectively. Oocyte maturation period of C. striata occurs from April to August. The highest gonads development was in July. Fecundity exhibited linear relationships with body length, body weight and ovarian weight.

Dry or wet? What is the best choice to determine gonadosomatic and hepatosomatic indexes in females of Macrobrachium amazonicum ?

The gonadosomatic (GSI) and hepatosomatic (HSI) indexes are widely used for the evaluation of reproductive biology in crustaceans. However, the use of either wet or dry weight for estimating these indexes is not standardized in the literature. Here, we compare the GSI and HSI indexes based on wet and dry samples of females of Macrobrachium amazonicum from populations with different phenotypes (large and small‐size prawns) as a model. In our analysis, for GSI, the application of wet weight tended to overestimate the values recorded in the initial (I‐II) and intermediate (III) stages of ovarian development, and underestimate the results in the final (IV‐V) stages when compared to the use of dry weight. This could be attributed to the reduction in cytoplasmic organelles and water content of the cytosol due to the increase in yolk and lipids in the oocytes throughout ovarian development. For HSI, no significant differences were detected between wet and dry samples. However, the dry HSI computed at all stages of development was overestimated when compared to the use of wet samples, and only the hepatopancreas of females at the final stage (V) showed a significant decrease in water content. Thus, our results indicate that M. amazonicum females maintain constant hepatopancreatic reserves throughout ovarian development. In this way, the use of dry weight is more adequate to estimate GSI and HSI in this species.

Integrated analysis of differentially expressed microRNAs and mRNAs to screen miRNAs and genes related to reproduction in Macrobrachium nipponense.

Female Macrobrachium nipponense has the characteristic of short sexual maturity during the breeding season, which can increase breeding risk and lead to prevalent female individual miniaturization. In this study, we characterized micro (mi)RNA-seq data of the eyestalk (E) and cerebral ganglia (B) of female M. nipponense during breeding and non-breeding seasons. A total of 393 and 189 differentially expressed miRNAs (DE miRNAs) were identified in BSE vs. NBSE and BSB vs. NBSB, respectively. The most abundant up- and down-regulated DE miRNAs were miR-124, miR-14, and miR-7. Enrichment analysis showed that DE miRNA target genes were mainly involved in 'metabolic process' and 'binding', and were associated with 'neurohormonal regulation' and 'photoreceptor activity' signaling pathways. Integrated analysis of miRNA-mRNA expression showed that the most abundant DE miRNAs were miR-14 and miR-278 in BSE vs. NBSE and BSB vs. NBSB, respectively. Four pairs of DE miRNAs and their corresponding target annotated genes were selected from the DE miRNA-mRNA interaction network (bmo-miR-316-5p/opsin protein, ame-miR-125/skeletal muscle actin 8, dmo-miR-278/sugar transporter, and tca-miR-3885-5p/5-HT1 receptor). Gene expression analysis of these four pairs in different ovary development stages showed their potential regulatory roles in ovary maturation.

Oocyte maturation and active motility of spermatozoa are triggered by retinoic acid in pen shell <i>Atrina pectinata</i>

For recovery of the declining population of pen shells in the wild, the production of pen shell juveniles for transplantation or aquaculture is underway in Japan. For more stable juvenile production, artificial fertilization methods for pen shells are needed, but methods to induce oocyte maturation (meiosis resumption) used in other bivalves, which make oocytes fertilizable, were ineffective for pen shells. Here, we report evidence showing that retinoic acid (RA) has strong activity in inducing oocyte maturation and activating sperm motility in pen shells. Treatment of fully developed oocytes with 1.0 μM all-trans-RA (at-RA) induced germinal vesicle breakdown, a typical morphological sign of oocyte maturation, but 1.0 μM at-retinol and at-retinal, 2 mM ammonia, and 1.0 μM serotonin were ineffective. Treatment with at-RA for 30 min was sufficient for oocyte maturation and was more potent than its isomers, 9-cis- and 13-cis-RA. Parallel results were obtained for sperm motility activation. Oocyte responsiveness to at-RA increased during the final stage of ovary development. Artificial fertilization was successful only with the oocytes treated with at-RA, and fertilized eggs developed to D-shaped (veliger) larvae without apparent morphological abnormalities. These results indicate the possible application of RA for the artificial fertilization of pen shells.

Oogenesis of Lithodes santolla: histological and histochemical characterization

Although Lithodes santolla is a resource with high commercial importance that has aroused interest in controlled reproduction in captivity, currently, little knowledge is available about basic reproductive aspects of this species. This research study describes detailed oogenesis stages at the histological level in adult females of the southern king crab of the Magellan and Chilean Antarctic Region. Oogenesis was quantitatively analyzed from the proliferation of 10-24 μm oogonia and during oocyte growth. Five stages of ovary development were identified: multiplication (0), previtellogenesis (I), vitellogenesis (II), maturity (III) and spawning (IV). Furthermore, 11 oocyte substages were distinguished: oogonia, chromatin nucleolus, early perinucleolus, late perinucleolus, oil globule, early vitellin globule, late vitellin globule, postvitelogenic, germinal vesicle migration and germinal vesicle breakdown. Primary vitellogenesis occurred in oocytes 180-185 μm, containing acidophilic globules and surrounded by a layer of thick follicle cells (20-40 μm). Secondary vitellogenesis was evident in oocytes at 315-321 μm with numerous acidophilic granules in the cytoplasm and surrounded by thin follicle cells; then, cortical crypts appeared, indicating the prematuration stage and preparation for ovulation. This study allows establishing gonadic changes that occur during a reproductive cycle of female L. santolla and help to strengthen aquaculture initiatives and management in the Magellan and Chilean Antarctic Region.

Ovarian transcriptome analysis of Mactra chinensis provides insights into genes expressed during the intermediate and ripening stages

Enhancing the production of aquatic animals is very important for fishery management and aquaculture applications. Ovaries have important functions in producing oocytes and hormones. The Chinese clam (Mactra chinensis) is a nutritious saltwater shellfish. Significant biochemical changes take place during the sexual maturation of M. chinensis; however, the genetic mechanisms of this process are unclear. Transcriptome sequencing can determine gene expression changes as development occurrs. In the present study, transcriptome sequencing was used to produce a comprehensive transcript dataset for the ovarian development of M. chinensis. The different ovarian developmental stages were determined using hematoxylin-eosin staining. There was identification of 54,172 unigenes at the intermediate stage and 63,081 at the ripening stage, and 80,141 all-unigenes were assembled to determine the molecular mechanism of ovarian development in M. chinensis. Quantitative real-time PCR for nine mRNAs confirmed the RNA-seq data. Functional annotation of the transcripts indicated there were important pathways in ovarian development, such as those involving the vitellogenin gene. Six pathways associated with ovarian development were identified: estrogen signaling pathway, GnRH signaling pathway, progesterone-mediated oocyte maturation, ovarian steroidogenesis, steroid hormone biosynthesis, and steroid biosynthesis. Significant upregulation of protein kinase alpha (PKA) and calmodulin (CAM) in four of the pathways indicates that PKA and CAM are active in M. chinensis ovarian development during maturation. Results of the present study provide the first comprehensive transcriptomic resource for M. chinensis ovaries, which will increase understanding of the molecular mechanisms underlying sexual maturation and promote molecular nutritional studies of M. chinensis.

Endoscopy and Cannulation as Non-Invasive Tools to Identify Sex and Monitor Reproductive Development in Arapaima gigas

The lack of tools for sex identification and assessment of gonadal development are hindering our ability to study the reproductive dysfunction of Arapaima gigas in captivity. This study initially aimed to validate a non-surgical endoscopy procedure to identify sex in juveniles and assess stage of ovary development in female broodstock under field operational conditions. Cannulation, assisted through the description of the genital anatomy, made ovarian biopsy possible to describe oocyte development from primary growth to pre-ovulation, providing a first classification scheme for oogenesis in the species including description of the micropyle morphology using scanning electron microscopy. Cannulation was also successfully performed without endoscopic guidance, which allowed monitoring of ovarian development along the reproductive season together with profiling of plasma sex steroids (17β-estradiol [E2] and 11-ketotestosterone [11-KT] in females and males, respectively). The monitoring of our study population showed females paired with males in earthen ponds sexually matured and reached oocyte maturation during the spawning season. However, since no spawning was recorded, eggs had either been resorbed or released and not fertilized by the male. Plasma E2 levels remained high in females, as expected in an asynchronous species during the spawning season with multiple batches of oocytes being recruited. Plasma 11-KT showed a tendency to decrease, suggesting a male reproductive dysfunction or the end of the reproductive season with a lack of synchronization between sexes. In conclusion, endoscopy and cannulation are tools that can be promptly applied to aid sex identification, assessment of reproductive function, and overall broodstock management in wild and captive stocks. These tools will greatly help future studies looking at the effects of environmental, social, and hormonal cues on reproductive development with the aim of developing a spawning induction protocol for the species.

Seasonal changes in hepatosomatic index, gonadosomatic index and plasma estradiol‐17β level in captively reared female rabbit fish ( Siganus guttatus )

The seasonal changes in hepatosomatic index (HSI), gonadosomatic index (GSI) and plasma estradiol‐17β (E₂) level in female rabbit fish (Siganus guttatus) were investigated. The relationship between plasma E₂ levels with these indices and ovarian growth was also evaluated. Each month, at least 10 female broodfish were sacrificed to collect liver, ovary and blood for HSI, GSI and plasma E₂, respectively. GSI and HSI were calculated as percentage (%) of relative weight of gonad and liver to total body weight, respectively. Plasma E₂ level was measured using enzyme‐linked immunosorbent assay method (ELISA). Ovaries were cut and stained for histological observation. The results included seasonal changes in plasma E₂ levels, stages of ovarian development, GSI and HSI. The highest level of E₂ was observed in June (1,445.62 pg/ml) and during vitellogenesis (2,305 pg/ml). GSI and HSI values significant fluctuated monthly. The highest HSI and GSI were 1.72% in May and 3.58% in June, respectively. The pattern of plasma E₂ levels showed a relationship with GSI and different stages of ovarian development. HSI was associated with ovarian stages. During vitellogenesis, the highest value (1.9%) of HSI was observed. Histological sections showed that rabbit fish is a multiple spawner. These results contribute to further understanding of female rabbit fish reproductive biology in captivity. Important reproductive parameters such as HSI, GSI and E₂ can be used to indicate maturation status of this fish species.

Identification and Characterization of the DMRT11E Gene in the Oriental River Prawn Macrobrachium nipponense.

The doublesex and mab-3 related transcription factor (DMRT) gene family involvement in sex development is widely conserved from invertebrates to humans. In this study, we identified a DM (Doublesex/Mab-3)-domain gene in Macrobrachium nipponense, which we named MniDMRT11E because it has many similarities to and phylogenetically close relationships with the arthropod DMRT11E. Amino acid alignments and structural prediction uncovered conservation and putative active sites of the DM domain. Real-time PCR analysis showed that the MniDMRT11E was highly expressed in the ovary and testis in both males and females. Cellular localization analysis showed that DMRT11E was mainly located in the oocytes of the ovary and the spermatocyte of the testis. During embryogenesis, the expression level of MniDMRT11E was higher at the cleavage stage than at other stages. During the different stages of ovarian development, MniDMRT11E expression gradually increased from OI to OIII and decreased to the lowest level at the end of OIV. The results indicated that MniDMRT11E probably played important roles in embryonic development and sex maturity in M. nipponense. MniDMRT11E dsRNA injection also significantly reduced vitellogenin (VG) expression and significantly increased insulin-like androgenic gland factor (IAG) expression, indicating a close relationship in gonad development.

Circulating miRNA Profiling of Women at High Risk for Ovarian Cancer

Survival of epithelial ovarian cancer patients remains poor without significant change over many decades. There is a need to better identify women at high risk (HR) for ovarian cancer. We propose that miRNA dysregulation may play critical roles in the early stages of ovarian cancer development. Circulating miRNAs may represent an important biomarker in this context, and miRNA profiling of serum in women at HR compared to those at low risk (LR) may give insights in tumor initiation pathways. There is also rationale for a specific focus on regulation of the androgen and its related hypoxia pathways in tumor initiation. We hypothesized that subsets of these pathway related miRNAs may be downregulated in the HR state. Serum from four HR and five LR women were sequenced and analyzed for 2083 miRNAs. We found 137 miRNAs dysregulated between the HR and LR groups, of which 36 miRNAs were overexpressed in HR and the vast majority (101 miRNAs or 74%) downregulated in the HR, when compared to LR serum. mRNA targets for the differentially expressed miRNAs were analyzed from three different miRNA-mRNA interaction resources. Functional association analysis of hypoxia and androgen pathway mRNA targets of dysregulated miRNAs in HR serum revealed that all but one of the miRNAs that target 52 hypoxia genes were downregulated in HR compared to LR serum. Androgen pathway analysis also had a similar expression pattern where all but one of the miRNAs that target these 135 identified genes were downregulated in HR serum. Overall, there were 91 differentially expressed miRNA-mRNA pairings in the hypoxia analysis. In the androgen-related analysis, overall, there were 429 differentially expressed miRNA-mRNA pairs. Our pilot study suggests that almost all miRNAs that are conserved and/or validated are downregulated in the HR compared to LR serum. This study, which requires validation, suggests that, via miRNA dysregulation, involvement of both hypoxia and its related androgen pathways may contribute to the HR state. This pilot study is the first report to our knowledge that studies circulating miRNA profiling of HR and LR women.

Elucidation of the role of farnesoic acid O-methyltransferase (FAMeT) in the giant freshwater prawn, Macrobrachium rosenbergii: Possible functional correlation with ecdysteroid signaling

Farnesoic acid O-methyltransferase (FAMeT) is a key enzyme involved in catalyzing the conversion of farnesoic acid (FA) to methylfarnesoate (MF) in the mandibular organ (MO) of crustaceans. In this study, a full-length cDNA encoding a 278-amino-acid FAMeT protein (MrFAMeT) was characterized from the giant freshwater prawn, Macrobrachium rosenbergii. Bioinformatics analysis revealed a high degree of conservation of FAMeT among crustaceans and a close phylogenetic relationship between MrFAMeT and that of Scylla paramamosain. The prokaryotic expressed MrFAMeT could catalyze the conversion of FA to MF in a radiochemical assay. Expression analysis by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that MrFAMeT mRNA was highly expressed in the muscle and the hepatopancreas of both females and males. During the molt cycle and the ovarian development, the mRNA expressions of MrFAMeT displayed stage-specific patterns in the muscle of both sexes and the female hepatopancreas, and the highest expressions were detected at intermolt and ovarian development stage V. Double stranded RNA (dsRNA)-mediated RNA interference (RNAi) of MrFAMeT increased expressions of myostatin in the muscle of both sexes and reduced expressions of vitellogenin (Vg) in the female hepatopancreas. Furthermore, both in the muscle and the hepatopancreas, silence of MrFAMeT downregulated the expression of ecydone receptor gene (MrEcR) and silence of MrEcR decreased the expression of MrFAMeT as well. Results in our study indicate that MrFAMeT is involved in prawn muscle growth and female vitellogenin biosynthesis and its function may be closely related with the ecdysteroid signaling.

Multiple vitellogenin genes (vtgs) in large yellow croaker (Larimichthys crocea): molecular characterization and expression pattern analysis during ovarian development.

The large yellow croaker (Larimichthys crocea) is a marine fish that is economically important to Chinese fisheries, and its reproductive and developmental biology have been extensively investigated. However, the molecular mechanism of oogenesis in L. crocea is not clear. Here, we investigated the multiple vitellogenin (Vtg) system in large yellow croaker. Three different vtg cDNA sequences, including vtgAa, vtgAb and vtgC, were cloned, which indicate the existence of multiple Vtg proteins in large yellow croaker (Lc-Vtgs). Subsequently, the vtg cDNA sequences and predicted Vtg protein structures were analysed, and Vtg protein structures were found to be highly conserved. To research the expression of vtgs during the development of the ovaries, we examined ovarian development and oogenesis by histological analysis. Four stages of ovary development - stages II, III, IV and V - were observed and their boundaries were defined. Soon afterwards, the expression of vtgs in the liver (known as the main site of Vtg synthesis in teleosts) and ovary were analysed. The expression of vtgs was detected in the two tissues. Interestingly, in the early stages of development (stages II and III), there is little or no generation of yolk granules and the expression of vtgs in the liver is low. However, in the late stages (stages IV and V), yolk granules are generated rapidly and the expression of vtgs is significantly increased in the liver. These results support the hypothesis that the Vtgs were synthetized by the liver, and absorbed by the growing oocytes to promote oogenesis in large yellow croaker. We also detected the presence of vtg mRNA in the liver cells and oocytes by in situ hybridization, which indicated that vths were expressed both in the liver and ovaries. Importantly, we found that the distribution of vtgAa and vtgAb mRNA was close to the sites of yolk granule formation in oocytes.

Comparative expression analysis of let-7 microRNAs during ovary development in Megalobrama amblycephala.

As a critical regulator of gene expression, let-7 family miRNAs have been reported to be involved in multiple physiological processes. In this study, in order to elucidate the putative regulatory effect of let-7 miRNAs during fish gonadal development and to identify which member is crucial for this regulation, the expression of ten members including let-7a/b/c/d/e/f/g/h/i/j were quantified in ovary, pituitary, and brain tissues during the different ovarian developmental stages of blunt snout bream Megalobrama amblycephala. According to the data from analysis of expression patterns, let-7a showed the highest expression value in almost all the tested ovaries, pituitaries, and brains, with let-7b and let-7d moderately expressed, following by other let-7 miRNAs. In terms of the differential expression levels of ten let-7 miRNAs at each developmental stage, the results showed that let-7a/b/d/f/h expression gradually increased during the ovary development from stage I to V and dropped significantly at the phase VI in ovary tissues. However, the expression of let-7a/b/e/f in pituitary increased during the ovary development from stage I to IV and declined at stage V. Among all the let-7 miRNAs, let-7a/b/d had the highest expression and their expression patterns were consistent with the gonad development of M. amblycephala. Furthermore, the mostly predicted target genes of let-7 miRNAs are significantly enriched in signaling pathways closely related to gonadal development through KEGG enrichment analysis. These results indicate that let-7 miRNA members, especially let-7a/b/d, may play important roles in the regulation of ovary development in M. amblycephala through negatively regulating expression of their target genes.

De novo transcriptome analysis of eyestalk reveals ovarian maturation related genes in Macrobrachium rosenbergii

Macrobrachium rosenbergii is a commercially important freshwater crustacean species cultured across the world. The economic benefit of M. rosenbergii industry is closely related with gamete quality which unfortunately is unstable in artificial breeding. Improving gamete quality might be achieved through a better understanding of ovarian maturation, but the molecular mechanism is still unclear. This study presented a transcriptome analysis of eyestalk between ovary undeveloped juvenile M. rosenbergii and ovary maturing adults to identify genes involved in ovarian maturation. Six cDNA libraries were constructed with three biological replicates in each group. More than 44 billion raw reads were obtained from each sequencing library using Illumina HiSeq X Ten sequencing platform and 53,878 unigenes were generated after de novo assembly. A total of 15,792 (29.3%) unigenes were annotated by searching against the public databases including NR, Swissprot, KOG, GO and KEGG databases. The comparative transcriptome analysis identified 4815 significantly differentially expressed genes (DEGs) between two groups in which 2961 genes were up-regulated and 1854 genes were down-regulated. Sixteen DEGs were identified to be candidate genes involved in ovarian maturation including neurohormones, neurotransmitter receptor, insulin-like peptides and related receptors, steroidal hormones, methyl farnesoate (MF) related genes and so on. The function of five genes was further verified using mRNA expression detecting at different ovarian developmental stages. Enrichment analysis revealed 65 significantly changed KEGG pathways, in which 2 pathways (insulin signaling pathway and TGF-beta signaling pathway) showed obvious association with ovarian maturation. Data from this study provided fundamental information about gene expression of eyestalk at different ovarian developmental stages in M. rosenbergii, which might be helpful for revealing the regulation mechanism of ovarian maturation and then further contribute to improving gamete quality in aquaculture.