Previtellogenic Oocytes Research Articles

In vivo exposure to pyriproxyfen causes ovarian oxidative stress and inhibits follicle maturation in zebrafish.

We analyzed the effects of pyriproxyfen (PPF) on oxidative stress and ovarian morphology in zebrafish. PPF (10-9 M) exposure increased reactive oxygen species generation in ovaries, in association with a decrease in glutathione content. The activities of glutathione S-transferase, superoxide dismutase, and catalase were increased, while γ-glutamyltransferase activity was not altered by pesticide treatment. The histology of ovarian tissue showed an increase in the number of previtellogenic oocytes I, and a decrease in the rate of vitellogenic oocyte (VIT) count, suggesting inhibition of follicular maturation. An increase in the thickness of the vitelline envelope was observed in VIT, as was a tendency toward an increase in atresia in the ovary of the PPF-treated group. These findings indicate that the deleterious effect of PPF on ovarian maturation is mediated by a redox imbalance and oxidative damage. So, PPF acts as an endocrine disruptor chemical and may compromise fish reproduction by reducing female fertility.

Gonadal proliferation and reproductive cycle of the exotic sea squirt Cnemidocarpa amphora () (Pleurogona, Styelidae) sampled for the first time from the northern coast of Arabian Gulf in Saudi Arabia.

Specimens of Cnemidocarpa amphora were collected monthly from the Arabian Gulf from September 2017 to August 2018. Parts of their gonads were prepared for histological testing. The gonads' diameters varied by month. Each gonad contained many ovarian follicles with different morphologies and was surrounded by several testicular follicles. The ovarian and testicular follicles were separate, although the latter were always present near the former. Repeated measures ANOVA tests were conducted to investigate monthly changes in oocyte stages. In squirts measuring 12-13 cm in length, the gonads measured 30-50 mm from July to August; 20-25 mm from September to October; 15-20 mm from November to February; and 25-40 mm from March to June. Oogonia budded from the germinal epithelium with diameters of 20-30 µm. Previtellogenic oocytes measuring 70-120 µm were characterized by the deposition of small granules of protein around the nucleus, a continuous layer of follicular cuboidal epithelium, and scattered vacuoles in the ooplasm. The measurement of gonads and oocyte diameters were performed by image analysis (Image scope 2.3, Image Line, Inc.) and stage micrometer. The vitellogenic oocytes measured 130-220 µm and the follicular epithelium consisted of flattened and cuboidal layers. Beneath the vitelline membrane, scattered test cells appeared in the ooplasm and different granules of protein and MPS were deposited in the ooplasm. In the later phase, lipid droplets began to appear in the ooplasm. Yolk bodies formed after the impregnation of various granules together and the oocyte was ready to be shed. Before spawning, a yolk membrane appeared above the ooplasm. Post-vitellogenic oocytes, in which the homogeneity of ooplasm was restored, underwent gradual lysis and entered the atretic phase. Different stages of sperm development were present year-round in different follicles of the same squirt; hence, the testes were always mature.

Reproductive and morphometric traits of freshwater mussel Lamellidens marginalis and associated hydrology in the Ratargul freshwater Swamp Forest, Bangladesh

A total of 300 freshwater mussels Lamellidens marginaliswere sampled from the Ratargul Freshwater Swamp between December 2020 and November 2021 to investigate the relationships between length, weight, soft tissue wet parameters, sex ratio, condition indices, gonadal histology, and hydrological features of swamp forest habitat. The shell length and shell weight varied between 5.00 and 9.50 cm and 12.26–78.96 g respectively. The length at maturity Lm was recorded as 5.98 cm and the calculated value of χ2 suggested a dominant female stock at the study site (P < 0.05). The relationship between different morphometrics ofLamellidens marginalisand environmental parameters exhibits a very weak regression coefficient value (P < 0.05), suggesting a weak effect of seasonality on the growth metrics. Previtellogenic and vitellogenic oocytes were observed in the ovary, and mature spermatids were found in the testes in samples collected in the rainy season.

Post-embryonic development of the female reproductive system of the Neotropical termite Silvestritermes euamignathus (Isoptera: Termitidae: Syntermitinae)

In termites, the maturation of the female reproductive system is progressive following post-embryonic development. In Silvestritermes euamignathus, the imaginal line is characterized by five nymphal instars that develop into the imago, but it can deviate in some instars to neotenic reproductives. In order to understand the rate of development of the female reproductive system throughout post-embryonic development, we analyzed the morphology of the reproductive system of nymphs, characterizing the stage of development and comparing it with neotenics and primary queens. We also followed embryonic development and compared eggs from neotenics and primary queens from incipient and mature colonies. Our results showed that gonadal development follows the post-embryonic instars, and previtellogenic oocytes are present in third-instar nymphs and are retained for the next two successive nymphal instars. The full maturation of the ovaries with vitellogenic oocytes requires molting to either imagos or neotenics. Eggs from neotenics follow the same embryonic development and each stage presents similar volume when compared to those of mature primary queens. Eggs of primary queens from incipient colonies are greater in volume than those of mature primary queens and of neotenics, suggesting an investment in egg quality rather than quantity during colony foundation.

Role of the Melanocortin System in Gonadal Steroidogenesis of Zebrafish.

Simple SummaryControl of reproduction in both males and females is complex, and a number of hormonal systems translate internal and/or external information to the hypothalamic-pituitary-gonadal (HPG) system. The control of reproduction integrates this high-energy demanding event to the internal (stored energy availability) and external (resources availability for progeny) conditions, thus increasing environmental fitting and offspring survival. In this paper, we describe new effects of the melanocortin system on gonadal physiology. Both adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormones (MSHs) modulate gonadal steroid secretion working throughout specific melanocortin receptors expressed in different gonadal cell types. The inhibitory effects of ACTH on gonadotropin-stimulated estradiol secretion seems to be related to the deleterious effects of stress on the female reproductive axis. On the contrary, the physiological involvement of MSH peptides on ovarian gametogenesis as well as the stimulatory effects of ACTH on testicular testosterone production remain unknown and further studies are required to understand melanocortin role on gonadal physiology.In teleost, as in other vertebrates, stress affects reproduction. A key component of the stress response is the pituitary secretion of the adrenocorticotropic hormone (ACTH), which binds to the melanocortin 2 receptor (MC2R) in the adrenal glands and activates cortisol biosynthesis. In zebrafish, Mc2r was identified in male and female gonads, while ACTH has been shown to have a physiological role in modulating reproductive activity. In this study, the hypothesis that other melanocortins may also affect how the zebrafish gonadal function is explored, specifically steroid biosynthesis, given the presence of members of the melanocortin signaling system in zebrafish gonads. Using cell culture, expression analysis, and cellular localization of gene expression, our new observations demonstrated that melanocortin receptors, accessory proteins, antagonists, and agonists are expressed in both the ovary and testis of zebrafish (n = 4 each sex). Moreover, melanocortin peptides modulate both basal and gonadotropin-stimulated steroid release from zebrafish gonads (n = 15 for males and n = 50 for females). In situ hybridization in ovaries (n = 3) of zebrafish showed mc1r and mc4r in follicular cells and adjacent to cortical alveoli in the ooplasm of previtellogenic and vitellogenic oocytes. In zebrafish testes (n = 3), mc4r and mc1r were detected exclusively in germ cells, specifically in spermatogonia and spermatocytes. Our results suggest that melanocortins are, directly or indirectly, involved in the endocrine control of vitellogenesis in females, through modulation of estradiol synthesis via autocrine or paracrine actions in zebrafish ovaries. Adult zebrafish testes were sensitive to low doses of ACTH, eliciting testosterone production, which indicates a potential role of this peptide as a paracrine regulator of testicular function.

Cloning and function of the cyclin‐dependent kinase 9 gene in ovarian development of the ridgetail white prawn, Exopalaemon carinicauda

To investigate the key genes affecting ovarian development in the ridgetail white prawn, Exopalaemon carinicauda, we cloned the full-length cDNA of the CDK9 gene and discussed its role in ovarian development. Our results show that the gene has an open reading frame of 1155 bp, encoded 383 amino acids, and it has a conserved cyclin binding motif S_TKc. The open reading frame of CDK9 was expressed as a 43.96 Kda molecular weight protein. The phylogenetic tree analysis showed that the CDK9 gene has the highest homology with the CDK9 genes of Penaeus vannamei and Penaeus japonicus. The results of qRT-PCR show that the CDK9 gene was also expressed in the eye stalk, stomach, heart, gill, intestine, muscle, ovary, hepatopancreas and ventral cord. The expression level was highest in the ovary, which was significantly different from the other tissues (p < 0.05). In the five stages of ovarian development, the expression level of the CDK9 gene was highest in stage I, which was significantly different from the other stages (p < 0.05). In situ hybridization tests showed strong expression signals in the membrane, cytoplasm and nucleus of oocytes and previtellogenic oocytes. Nevertheless, following the proliferation of oocytes, the expression signals became much stronger in the oocyte membrane and in the whole follicular cells located in the interstitial space. The RNA interference results show that the expression of the CDK9 gene significantly affects the expression of the Vg and VgR genes. Our findings suggest that CDK9 plays an important role in the synthesis and transportation of vitellin.

Sex steroid profile during oocyte development and maturation in the intertidal worm Marphysa madrasi (Polychaeta: Eunicidae) from the east coast of India

Marphysa madrasi is a commercially valuable maturation diet in crustacean aquaculture. This study presents the first detailed investigation of oogenesis in the intertidal polychaete worm M. madrasi and reports the steroid profile during oocyte growth and development. Oogenesis is extraovarian type I, originating from coelomic epithelial cells, with four stages of development - primary growth, early vitellogenic, late vitellogenic, and maturation. The primary growth phase contains oogonial cells and previtellogenic oocyte clusters in the early, mid, and late stages of development form a dispersed ovary attached to blood vessels. The late previtellogenic oocytes detach from the ovary at the onset of vitellogenesis. The detached oocytes complete vitellogenesis and final maturation in the coelomic fluid as solitary free-floating cells without any connection with follicle cells. The worms display asynchronous reproduction with a heterogeneous population of developing oocytes. Steroid extracts from the polychaete homogenates in different stages of oogenesis were identified by HPLC and confirmed by LC-MS/MS. In M. madrasi, two vertebrate-type steroids, pregnenolone (P5) and 17α-hydroxyprogesterone (17-OHP) were detected and quantified. The P5 levels were low in immature worms but increased significantly by ∼ 8.3-fold in the previtellogenic stage and peaked during oocyte maturation. 17-OHP levels were low in immature worms but gradually increase as the oogenesis progress to the primary growth and early vitellogenic phase, with a significant increase (p < 0.001) during the late vitellogenic phase. Although an increase in the concentration of P5 and 17-OHP during vitellogenesis and maturation of oocytes points to a possible role in reproduction, the absence of other vertebrate-type steroids in the investigated polychaete signifies a plausible uptake of P5 and 17-OHP from the environment.

Ovarian development of the crabEriphia gonagra(Fabricius, 1781) (Decapoda: Brachyura: Eriphiidae) in northeastern Brazil

AbstractWe describe the development and maturation of the germ cells of the brachyuran crab Eriphia gonagra (Fabricius, 1781). Monthly collections were conducted in Pernambuco state, northeastern Brazil, from November 2018 to October 2019 during the low spring tide. The anatomy of the reproductive system of female individuals follows the basic brachyuran pattern. Five cell developmental stages were identified based on the morphological changes in the developmental stages: oogonia (7.10 ± 1.85 µm), pre-vitellogenic oocytes (42.23 ± 8.61 µm), oocytes in initial vitellogenesis (130.49 ± 21.93 µm), vitellogenic oocytes (139.68 ± 23.45 µm), and mature oocytes (214.77 ± 90.81 µm). Developmental stages were defined and related to gonad coloration and germ- cell development; five maturational stages were identified: immature, initial maturation, advanced maturation, mature, and spawning/resting.

Expression and regulation of 42Sp50 in spotted scat (Scatophagus argus).

42Sp50 is an isoform of the eukaryotic translation elongation factor 1 A (eEF1A) and is vital for fish ovarian development. Spotted scat (Scatophagus argus) is a popular marine cultured fish species in Southern Asia and China, and its artificial reproduction is complicated, with a relatively low success ratio in practice. In this study, the 42Sp50 gene was cloned from spotted scat. Tissue distribution analysis showed that 42Sp50 was mainly expressed in the ovary. qRT-PCR showed that 42Sp50 expression levels gradually decreased insignificantly in the ovaries from phase II to IV. Western blot analysis showed that 42Sp50 was highly expressed in the ovary, while it was almost undetectable in the testis. Immunohistochemistry analysis stained 42Sp50 mainly in the cytoplasm of the previtellogenic oocytes in ovaries of normal XX-female and sex-reversed XY-female. Aside from fish and amphibians, 42Sp50 was also identified in some reptile species using genomic database searching. Analyses of the transcriptome data from four different fish species (Hainan medaka (Oryzias curvinotus), silver sillago (Sillago sihama), Nile tilapia (Oreochromis niloticus), and Hong Kong catfish (Clarias fuscus)) revealed ovaries biased expression of 42Sp50 in all, similar to spotted scat. While the neighbor genes of 42Sp50 did not show ovary biased expression in the fish species analyzed. Bisulfite Sequencing PCR (BSP) results showed that the DNA methylation level of 42Sp50 promoter was low in ovaries, testes, and muscles. The luciferase reporter assay demonstrated that Dmrt4 activated 42Sp50 expression in the presence of Sf1 or Foxh1. These results suggest that 42Sp50 may be involved in regulating the early phase oocytes development of spotted scat.

Male and female reproductive systems of the pseudocycnid copepod Cybicola armatus (Bassett-Smith, 1898) (Siphonostomatoida) infecting the commercially important scombrid fish Scomberomorus commerson: a light and electron microscopic study

Abstract Cybicola armatus (Bassett-Smith, 1898), a host-specific parasitic copepod, massively infects gill filaments of the scombrid fish, Scomberomorus commerson. While the female attaches to the host fish’s gills, the relatively small-sized male prefers to attach to the female genital somite in copulatory position. To our knowledge this is the first report on the structural features of male and female reproductive organs of C. armatus, a pseudocycnid parasite, using morphological, histological, histochemical and ultrastructural parameters. In males, the sac-like testis is followed by a highly coiled vas deferens ending with a bulbous spermatophore sac in which the round-shaped spermatozoa are abundantly seen. Histologically, germ cells in the testis are at different maturation stages and region-specific. In females, the ovaries are comprised of germarium, previtellogenic oocytes and follicle cells. However, interestingly, mature vitellogenic oocytes enveloped by follicular cells are seen only in the oviducts, which are ensheathed by longitudinal and circular muscle bands. The spermathecae of the post-copulatory females contain filiform spermatozoa, while in pre-copulatory specimens, the spermathecae appear as thin, flabby structures. The post-copulatory female carries spermatophore sacs at her genital orfice. The developing eggs in the egg sacs did not display synchrony with respect to stages of embryogenesis, as borne out from histological studies. The currently reported study also demonstrates, for the first time, the copulatory events of C. armatus that help us understand the mode of sperm transfer in this fish parasite. The total of information derived from the currently reported study is pertinent not only from the perspective of understanding the reproductive mechanism and adaptive strategies of a potential, but rather unexplored pseudocycnid copepod. Rather, it throws light as well on the necessity and, until now, inadequacy of investigations on parasitic crustaceans that inflict heavy damage to fisheries and contaminate the aquatic ecosystem.

Medaka, Oryzias latipes, egg envelopes are created by ovarian-expressed ZP proteins and liver-expressed choriogenins

The medaka (Oryzias latipes) egg envelope (chorion) is composed of three major glycoproteins, Zona Interna (ZI)-1, -2, and -3, that originate in the spawning female liver as the precursor proteins Choriogenin (Chg.)H, Chg.Hm, and Chg.L, respectively. These ZI and Chg. proteins contain a structural ZP protein domain that is conserved among the egg envelope proteins of all animals. While ovarian expression of ZP proteins (e.g., ZPCs and ZPB) has been reported in medakas, the functions of these proteins remain unknown. Thus, the present study aimed to determine whether the ovary-expressed medaka ZP protein, mZPC5, is involved in forming the chorion matrix.The mZPC5 gene (mzpc5) was expressed in the ovaries but not the livers of mature female medakas, as shown by reverse transcription-polymerase chain reaction assays with mzpc5-specific primers. In situ hybridization analysis revealed that ovarian mzpc5 expression was restricted to the ooplasm of early (stage I–III) previtellogenic oocytes, and its expression signal weakened with oocyte growth. Following sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis with anti-mZPC5 antibodies, two immunoreactive proteins were detected in the ovary and chorion extracts. These proteins were approximately 50 and 74 kDa in size, like ZI-3 and ZI-2, respectively.Immunohistochemical assays using anti-mZPC5 and anti-Chg.H antibodies localized the mZPC5 protein in the ooplasm of early previtellogenic oocytes. With oocyte growth, mZPC5 tended to accumulate in the chorion, co-localizing with Chg.H.We previously showed that ovary-expressed ZP proteins could not compensate for Chg.L function loss in gene knock-out (chg.l -/-) medakas. As in our previous study, the chg.l-/- females produced oocytes with thin chorions, resulting in infertile soft eggs. However, in the present study, mZPC5 and Chg.H were co-localized in the chg.l-/- chorions. These results suggested that in the medaka previtellogenic oocyte, 1) mZPC5 is secreted from the ooplasm and deposited on the outer surface of its plasma membrane, creating the thin chorion layer; and 2) following the accumulation of liver-derived Chgs., the 3D structure of the chorion matrix is formed cooperatively with mZPC5 and Chgs. during oogenesis. More research is needed to confirm the functions of mZPC5 in chorion structure and physiology.

Ovarian maturation of Penaeus subtilis (Decapoda: Penaeidae): A new insight to describe oocyte development and somatic structures

AbstractWe observed the presence of follicular cells (FC) in the ovaries of Penaeus subtilis (n = 1198), which led us to classify the development of germ cells into six phases: oogonia, previtellogenic oocytes, primary and secondary vitellogenic oocytes, mature oocytes and atretic oocytes. The FC changes their shape according to the development of germ cells and showed a different distribution along the ovary, which allowed differentiating vitellogenic oocytes into primary and secondary. We also observed that the postovulatory follicles (POF) are composed of follicular cells. The presence of POF in penaeids ovaries is rarely reported, but allows the differentiation between spent and resting stages, commonly grouped in reproductive biology research. Furthermore, observation of ovarian lining was useful to differentiate immature females from females that had spawned at least once. Thus, ovarian development was classified into six stages: immature, early developing, advanced developing, ripe, spent and resting. The distribution and shape variations of FC, ovarian lining features and presence of POF were considered crucial for the classification of ovarian maturation stages. The methods developed here may improve estimates of their reproductive cycle, size at first maturity and spawning season, which are important variables in future studies of the reproductive dynamics.

Aquatic Pollution and Risks to Biodiversity: The Example of Cocaine Effects on the Ovaries of Anguilla anguilla.

Simple SummaryThe increasing consumption of illicit drugs by man has determined the presence of these drugs in waters, both fresh and marine, in almost all the world. Many studies have shown the dangerousness of these substances to aquatic organisms, so the goal of this study was to investigate the effects of cocaine, one of the most used drugs, on the ovaries of the Anguilla anguilla eel, a critically endangered edible fish, to understand if this drug could also compromise fish reproduction. Our results show that cocaine may impair reproduction in eels through changes in oocyte morphology, expressions of enzymes crucial for oogenesis, and alteration in the serum levels of cortisol and gonadotropins, which regulate the development and maturation of eel gonads. Though this study was conducted on eels, other species may also suffer similar effects from cocaine exposure; our results, therefore, show that illicit drug use is a serious problem not only for humans but also for the environment.Pollution is one of the main causes of the loss of biodiversity, currently one of the most important environmental problems. Important sources of aquatic pollution are illicit drugs, whose presence in waters is closely related to human consumption; their psychoactive properties and biological activity suggest potential adverse effects on non-target organisms, such as aquatic biota. In this study, we evaluated the effect of an environmentally relevant concentration of cocaine (20 ng L−1), an illicit drug widely found in surface waters, on the ovaries of Anguilla anguilla, a species critically endangered and able to accumulate cocaine in its tissues following chronic exposure. The following parameters were evaluated: (1) the morphology of the ovaries; (2) the presence and distribution of enzymes involved in oogenesis; (3) serum cortisol, FSH, and LH levels. The eels exposed to cocaine showed a smaller follicular area and a higher percentage of connective tissue than controls (p < 0.05), as well as many previtellogenic oocytes compared with controls having numerous fully vitellogenic and early vitellogenic oocytes. In addition, the presence and location of 3β-hydroxysteroid dehydrogenase, 17β-hydroxysteroid dehydrogenase, and P450 aromatase differed in the two groups. Finally, cocaine exposure decreased FSH and LH levels, while it increased cortisol levels. These findings show that even a low environmental concentration of cocaine affects the ovarian morphology and activity of A. anguilla, suggesting a potential impact on reproduction in this species.

Morphology of female reproductive system of Mediterranean flatheaded peachborer, Capnodis tenebrionis (Linnaeus, 1761) (Coleoptera: Buprestidae)

AbstractCapnodis tenebrionis causes damage in many species of Rosaceae. The present study investigates on the morphology of the female reproductive system of C. tenebrionis. The female reproductive system of C. tenebrionis has a pair of ovaries, lateral oviducts, a common oviduct, spermatheca, and bursa copulatrix. Each ovary in C. tenebrionis consists of approximately 24 telotrophic meroistic type ovarioles. The ovarioles of C. tenebrionis have four regions (terminal filament, tropharium, vitellarium, and pedicel). Tropharium have trophocytes, young oocytes, and prefollicular cells. Vitellarium consists of previtellogenic, vitellogenic, and choriogenic oocytes. Previtellogenic oocyte is surrounded by cylindrical epithelial cells. Its ooplasm is homogeneous and basophilic. In vitellogenic oocyte, there are intercellular spaces between monolayered follicle cells. Its ooplasm has yolk granules and lipid droplets. Choriogenic oocyte are surrounded by chorion and single‐layered cylindrical cells. There are yolk granules and lipid droplets in its ooplasm which is asidophilic. In C. tenebrionis female, spermatheca and bursa copulatrix wall is surrounded by thin cuticular intima, monolayer epithelial, glandular cells, and muscle layer. Spermatheca lumen contains a large number of spermatozoa. Bursa copulatrix lumen is filled with secretory material. This study may be useful in terms of the morphology of mature female reproductive organs of Buprestidae and other coleopteran species.

Effects of gonadotropins, 11-ketotestosterone, and insulin-like growth factor-1 on target gene expression and growth of previtellogenic oocytes from shortfinned eels, Anguilla australis, in vitro.

Pituitary gonadotropins, metabolic hormones, and sex steroids are known factors affecting the advanced stages of ovarian development in teleost fish. However, the effects of these hormones and of the interactions between them on the growth of previtellogenic ovarian follicles are not known. In order to address this void in understanding, previtellogenic ovarian fragments from eel, Anguilla australis, were incubated in vitro with recombinant Japanese eel follicle-stimulating hormone (rec-Fsh), human chorionic gonadotropin (hCG), or 11-ketotestosterone (11-KT) in the presence or absence of recombinant human insulin-like growth factor-1 (IGF1). The results of long-term in vitro culture (21days) demonstrated that rec-Fsh and 11-KT, rather than hCG, caused significant increases in the diameter of previtellogenic oocytes. Meanwhile, only 11-KT induced a significant increase in lipid accumulation. Moreover, a greater effect on oocyte growth was observed when IGF1 supplementation was combined with 11-KT rather than with rec-Fsh or hCG. For short-term culture (24h), treatment with 11-KT in the presence or absence of IGF1 had no significant effects on mRNA levels of target genes (lhr, cyp19, cyp11b, lpl, and ldr) except for upregulation of fshr. There were no significant effects of rec-Fsh on expression of any target gene, whereas hCG downregulated the expression of these genes. There was no evidence for any interaction between the gonadotropins and IGF1 that resulted in growth of previtellogenic oocytes. Taken together, these results suggest that hormones from both the reproductive and the metabolic axes regulate the growth of previtellogenic oocytes in Anguilla australis.

Characterization, expression and function of the pyrokinins (PKs) in the giant freshwater prawn, Macrobrachium rosenbergii.

Pyrokinins (PKs) are neuropeptides that have been found to regulate a variety of physiological activities including reproduction in various insect and crustacean species. However, the reproductive roles of PKs in the giant freshwater prawn, Macrobrachium rosenbergii, have not yet been investigated. In this study, we identified the MroPK gene from next-generation sequence resources, which encodes a MroPK precursor that shares a high degree of conservation with the C-terminal sequence of FxPRLamide in other arthropods. MroPK is expressed within most tissues, except the hepatopancreas, stomach and gill. Within developing ovarian tissue, MroPK expression was found to be significantly higher during the early stages (stages 1-2) compared with the late stages (stages 3-4), and could be localized to the oogonia, previtellogenic and early vitellogenic oocytes. A role for PK in M. rosenbergii reproduction was supported following experimental administration of MroPK to ovarian explant cultures, which led to an increase in the production of progesterone and estradiol and upregulation of expression of steroidogenesis-related genes (3β-HSD and 17β-HSD) and vitellogenin (Vg). Together, these results support a role for MroPK in regulating ovarian maturation via steroidogenesis.

Dynamics of ovarian maturation during the annual reproductive cycle of the endangered fish Steindachneridion parahybae (Siluriformes: Pimelodidae) in captivity.

To characterize the female reproductive biology of the endangered species Steindachneridion parahybae in captivity, the authors used the concentration of gonadal steroids and the oocyte development during the annual reproductive cycle (RC) and after artificial induced spawning (AIS) until 48 h. Three stages of gonadal maturation were identified, based on morphological and physiological features: early maturation or previtellogenic (PRV) oocyte, advanced maturation or vitellogenic (VTG) oocyte and regression (REG) or follicular atresia. They identified and characterized the following stages of germ cells: oogonia, perinucleolar and cortical alveoli, and VTG and atretic oocytes during RC. The oestradiol levels were higher in PRV than those in VTG and REG during the RC, whereas androgens showed higher levels of oestradiol in VTG than those in PRV and REG. The progestogen levels remained unchanged during the whole RC. The final oocyte maturation (FOM) was achieved after AIS and postovulatory follicles (POF) were identified. Plasma concentration of progestogens (17α,20β-dihydroxy-pregnen-3-one and 17α-hydroxyprogesterone) increased considerably after AIS, remaining high up to 6h after AIS, and progressively decreased over time after AIS. During RC, the lack of FOM and POFs reveals that captivity negatively impacts S. parahybae reproduction. Nonetheless, the VTG stage of oocytes, reached during RC, is suitable for ovulation induction with artificial hormone manipulation, enabling the reproduction of this species in captivity and being essential for the success of fish farming and/or fish conservation programmes (conservationist aquaculture).

Cloning of gonadotropin Gph-alpha, FSH-beta and LH-beta subunits and seasonal profiles of steroid hormones in wild-caught Nile perch, Lates niloticus

The Nile perch (np; Lates niloticus) is a freshwater teleost species with a potential for aquaculture in freshwater surroundings. However, wild-caught breeders have persistently failed to spawn spontaneously in captivity. Cloning of the gonadotropin subunits and analysing seasonal variation in reproductive hormone levels for a 1-year period were done to gain knowledge on the physiological basis underlying the reproductive biology of np. The β-follicle-stimulating hormone (FSH-β) and β-luteinizing hormone (LH-β) subunits and their common α-glycoprotein (Gph-α) subunit were cloned using 3′ and 5′ RACE-PCR. The nucleotide sequences of the npgph-α, npfsh-β, and nplh-β subunits were 664, 580 and 675 nucleotides in length, encoding peptides of 124, 120 and 148 amino acids, respectively. The deduced amino acid sequence of each mature subunit showed high similarity with its counterparts in other teleost. Sequence analysis showed that npFSH-β is more similar to higher vertebrate FSH-βs than to higher vertebrate LH-βs. Heterologous immunoassay was calibrated to analyse pituitary LH levels. While the LH immunoassay showed parallelism of npLH with that of tilapia (ta), no parallelism for FSH was found. Levels of pituitary LH were higher in females at gonadal stages of vitellogenic oocytes, mature secondary oocytes and mature tertiary oocytes with migrating nucleus than in pre-vitellogenic oocytes and early and late perinucleolus oocytes. Using competitive steroid ELISA, variations in the levels of the steroid hormones 11-ketotestosterone (11-KT) in males and E2 in females were characterized in relation to month and reproductive index of Nile perch. Our findings show that in females, gonadosomatic index and plasma E2 were highly correlated (R2 = 0.699, n = 172) and peaked from September to November while in males, the gonadosomatic index and plasma 11-KT peaked from October to November. In female fish, both steroid hormones were detected in the plasma but greatly varied in concentrations. E2 in particular, increased with the developmental stage of the gonads. The levels of steroid hormones, E2 and 11-KT in females and males respectively increased with fish size (total lengths) and suggest that females mature at a body length of 40–59 cm than their counter part males that mature at a total length of 60–70 cm. Taken together, we describe seasonal endocrine differences in wild-caught adult Nile perch which could potentially be exploited to manipulate the reproductive axis in cultured breeders.

Identification and characterization of a new germline-specific marker vasa gene and its promoter in the giant freshwater prawn Macrobrachium rosenbergii.

Vasa gene encodes a protein member of DEAD-box superfamily of ATP-dependent RNA helicases, which plays a key role in germline development in metazoans. In present study, we identified a new germline-specific marker Mrvasa in the prawn Macrobrachium rosenbergii, whose genomic DNA sequence consists of 14 exons and 13 introns. A 2516bp of full-length Mrvasa cDNA encodes a protein of 603 amino acids. It contains nine conserved motifs, a zinc-finger motif, and RGG repeats. RT-PCR indicated that Mrvasa mRNA was specifically expressed in gonads. QPCR analysis further revealed that the expression of Mrvasa mRNA is much higher in testis than in ovary. In testis, the relative expression level of Mrvasa mRNA in late developing stage is significantly higher than that in early-middle developing stage. During ovarian development, no significant difference in expression was found. In situ hybridization demonstrated that Mrvasa mRNA was localized in germline cells including spermatogonia, spermatocytes, and spermatozoa in testes, and previtellogenic and vitellogenic oocytes in ovary. We then isolated the Mrvasa promoter and determined the transcription core region of this promoter. This is the first report on identification of vasa core promoter in crustaceans. Our results will provide a useful germline-specific marker Mrvasa for tracing germline cell formation and development in M. rosenbergii.

Oocyte growth, gonadosomatic index, hepatosomatic index and levels of reproductive hormones in goldspot mullet Planiliza parsia (Hamilton, 1822) reared in captivity

Goldspot mullet Planiliza parsia (Hamilton, 1822) is a commercial food fish in South-east Asian countries. Availability of hatchery produced seed is the major bottleneck in the development of aquaculture of this species. Therefore, an attempt was made to develop captive broodstock of P. parsia and understand its reproductive maturation. Broodstock reared for eighteen months were sampled and six maturation stages were defined (I-VI) in both the sexes. Ovarian biopsy, gonad histology, gonadosomatic index (GSI), hepatosomatic index (HSI) and reproductive hormones assay were performed in different maturation stages. Diameter of pre-vitellogenic, vitellogenic, post-vitellogenic and ripe oocytes was less than 369.80, 444.38, 477.41 and 665.5 µm, respectively. In the female, the highest value (p&lt;0.05) of GSI (18.31±1.15), HSI (1.84±0.84), 17 β-estradiol (E2) (898.45±0.35 pg ml-1), follicle stimulating hormone (FSH) (81.14±40.48 ml U ml-1) and vitellogenin (Vtg) were found at stage IV, whereas luteinising hormone (LH) attained its peak at V stage. In males, the concentration of testosterone (T) increased gradually from stage I (7.60±0.43 pg ml-1) to VI (40±2.89 pg ml-1). Histological observations revealed synchronous type of oogenesis and cystic type of spermatogenesis. The results of this study will help in the captive seed production of this species.Keywords: Captive maturation, Histology, Ovarian stages, Reproductive hormones, Vitellogenin