Ovaries Of Fish Research Articles

Expression and functional analysis of cathepsin L1 in ovarian development of the oriental river prawn, Macrobrachium nipponense

Cathepsin L is an important member of the cysteine protease family and has a central role in the development of ovaries in insects and fish. In this study, we analyzed the characteristics of the cathepsin L1 gene in the female oriental river prawn, Macrobrachium nipponense. The cathepsin L1 cDNA was 1173 bp in length, including a 960 bp open reading frame that encoded 319 amino acids. The cathepsin L1 gene was classified into the cathepsin L group and named Mn-CTS L1 by phylogenetic analysis. qRT-PCR analysis indicated that Mn-CTS L1 was highly expressed in the hepatopancreas and ovaries of female prawns. During the different stages of ovarian development, Mn-CTS L1 expression in the hepatopancreas and ovaries peaked before ovarian maturation. Mn-CTS L1 expression was higher at the post-larval stage of day 15 than at other stages of embryogenesis. In situ hybridization studies revealed that Mn-CTS L1 was localized in the oocyte of the ovary. Injection of Mn-CTS L1 dsRNA significantly reduced Mn-CTS L1 expression and significantly increased the expression of vitellogenin. Changes in the gonad somatic index also confirmed the inhibitory effects of Mn-CTS L1 dsRNA. These results suggest that Mn-CTS L1 has a key role in ovarian development.

Determination of Elemental Composition in Soft Biological Tissue Using Laser Ablation Inductively Coupled Plasma Mass Spectrometry: Method Validation.

Determination of elemental concentrations in biological tissue is fundamental to many environmental studies. Analytical methods typically used to quantify concentrations in such studies have minimum sample volumes that necessitate lethal or impactful collection of tissues. Laser ablation inductively coupled mass spectrometry (LA-ICP-MS) has small sample volume requirements and offers environmental practitioners an opportunity to employ low-impact sample collection methods. Environmental applications of LA-ICP-MS are limited by the lack of validated methods, partly due to the need for dry samples and scarcity of matrix-matched certified reference materials (CRMs). This study validates an LA-ICP-MS method to determine concentrations of 30 elements in soft biological tissue (fish ovary and muscle). Tissue samples (median: 0.48 grams (g); inter-quartile range: 0.30 g to 0.56 g wet weight) were dehydrated, powdered, compressed into pellets (weighing approximately 0.03 g) and analyzed using LA-ICP-MS alongside three matrix-matched CRMs. The method yielded concentration determinations for CRM elements that were typically accurate to within 30% of theoretical concentrations, and precise (relative standard deviation <20%). These results were repeatable: accuracy rarely deviated from theoretical values by more than 20%, and precision rarely exceeded 33%. Determinations for biological samples were replicable irrespective of tissue (ovary or muscle). There was good linearity between analyte signal strength and theoretical concentration (median R2 ≥ 0.981 for all elements) across ranges typically encountered in environmental studies. Concentrations could not be consistently obtained (i.e., determined concentrations were typically below detection limits) for boron, vanadium, molybdenum, and cadmium in muscles, and arsenic in both ovaries and muscles; however, detection limits were sufficiently low for most environmental contexts. Further methodological refinement could include the incorporation of spiked standards to extend linear ranges, and fine-tuning instrument parameters to obtain smoother signal intensities for rare elements. The method presented promotes the use of low-impact sample collection methods while enabling high-quality determinations of elemental concentrations in biological tissues.

A central role for cAMP/EPAC/RAP/PI3K/AKT/CREB signaling in LH-induced follicular Pgr expression at medaka ovulation†.

Nuclear progestin receptor (PGR) is a ligand-activated transcription factor that has been identified as a pivotal mediator of many processes associated with ovarian and uterine function, and aberrant control of PGR activity causes infertility and disease including cancer. The essential role of PGR in vertebrate ovulation is well recognized, but the mechanisms by which PGR is rapidly and transiently induced in preovulatory follicles after the ovulatory LH surge are not known in lower vertebrates. To address this issue, we utilized the small freshwater teleost medaka Oryzias latipes, which serves as a good model system for studying vertebrate ovulation. In the in vitro ovulation system using preovulatory follicles dissected from the fish ovaries, we found that inhibitors of EPAC (brefeldin A), RAP (GGTI298), PI3K (Wortmannin), AKT (AKT inhibitor IV), and CREB (KG-501) inhibited LH-induced follicle ovulation, while the PKA inhibitor H-89 had no effect on follicle ovulation. The inhibitors capable of inhibiting follicle ovulation also inhibited follicular expression of Pgr and matrix metalloproteinase-15 (Mmp15), the latter of which was previously shown to not only be a downstream effector of Pgr but also a proteolytic enzyme indispensable for follicle rupture in medaka ovulation. Further detailed analysis revealed for the first time that the cAMP/EPAC/RAP/PI3K/AKT/CREB signaling pathway mediates the LH signal to induce Pgr expression in preovulatory follicles. Our data also showed that phosphorylated Creb1 is a transcription factor essential for pgr expression and that Creb1 phosphorylated by Akt1, rather than PKA, may be preferably used to induce pgr expression.

Changes in Cadmium Concentration in Muscles, Ovaries, and Eggs of Silver European Eel (Anguilla anguilla) during Maturation under Controlled Conditions.

Simple SummaryThe European eel (Anguilla anguilla) is currently a critically endangered fish species (IUCN list). This is thought to be due to many factors, including pollution of the aquatic environment with heavy metals, especially cadmium (Cd) and mercury (Hg), which may have a negative impact on the condition and abundance of the European eel population. Owing to bioaccumulation, the level of cadmium in the tissues of fish, especially those at the top of the trophic chain, may be significant and endanger the health and effective spawning success of the species due to impairment of the reproductive capacity. In this study, it was found that female eels’ muscles contain significantly less cadmium than their ovaries and eggs. This indicates the transfer of cadmium from the maternal organism to the offspring organism.This study determined the contents of cadmium (Cd) in the muscles, ovaries, and eggs of silver female European eels. The analysis of cadmium content was performed on female European eels caught during commercial fishing in freshwater in Warmia and Mazury (Poland), and then subjected to artificial maturation and ovulation processing under controlled conditions. The content of cadmium (Cd) in the tissues was determined by flameless atomic spectrometry using an electrothermal atomizer. The analysis showed statistically significant differences between the cadmium content in the muscles, ovaries, and eggs (p < 0.05) of female European eels. The lowest cadmium content was found in the muscle tissue (0.0012 ± 0.0001 mg kg−1 wet weight) and the highest in eggs (after ovulation) (0.0038 ± 0.0007 mg kg−1 wet weight). Moreover, a relationship was found between the cadmium content in the muscle tissue and the ovaries (R = 0.673; p = 0.0117) in the same fish. The movement of cadmium from tissues to oocytes may indicate a significant problem concerning this heavy metal content in the reproduction of European eel.

Histopathological changes in Oreochromis mossambicus (Peters, 1852) ovaries after a chronic exposure to a mixture of the HIV drug nevirapine and the antibiotics sulfamethoxazole and trimethoprim

The burden of the human immunodeficiency virus and acquired immunodeficiency syndrome (HIV/AIDS) infection has transformed the African continent into a major consumer of antiretrovirals (ARVs) drugs. In addition to HIV burden, the African continent has also a high incidence of tuberculosis (TB) and has been experiencing recurring outbreaks of several other viral, bacterial, and parasitic epidemic diseases. The novel severe acute respiratory syndrome coronavirus 2 (SARS-COV-2 or Covid-19) pandemic outbreak is adding to the continent’s infectious diseases burden as experts are predicting that it will be here for a long time. One of the consequences of these infectious diseases is that antiviral and antibiotic compounds have become some of the most consumed pharmaceuticals on the continent. Many of these drugs have been frequently detected in surface waters across Africa. There is limited information available on the adverse effects of the mixtures of different types of pharmaceuticals in African aquatic environments on fish reproduction. The present study investigated the effects of the ARV drug nevirapine (NVP - 1.48 and 3.74 μg/L) and its mixture with the antibiotic sulfamethoxazole (3.68 μg/L) and trimethoprim (0.87 μg/L) on O. mossambicus gonads using histopathological endpoints as biomarkers. The fish (n = 52) were exposed for 30 days in a static renewal system. Female O. mossambicus exposed to nevirapine (3.74 μg/L) and to NVP – antibiotic mixture recorded higher ovary indices. Statistically significant differences were found in female ovary indices between the fish exposed to NVP (3.74 μg/L) and the control fish (p = 0.002) as well as between the fish exposed to the NVP - antibiotic mixture and the control fish (p = 0.009). The main observed histopathological changes in the ovaries were increased vitellogenic oocyte atresia and vacuolation of the interstitial tissue in the fish exposed to NVP - antibiotic mixture. It is evident that the presence of NVP - antibiotics mixture in water triggered the observed histopathology in female fish ovaries. The detected abnormal high rate of atretic oocytes could result in impaired fish reproduction.

Turnover rates for muscle, mucus and ovary tissues of ayu fish (Plecoglossus altivelis altivelis) in multiple stages determined through carbon and nitrogen stable isotope analyses

AbstractStable isotope ratios are powerful indicators that facilitate the understanding of the diet shifts and between‐habitat migration of aquatic animals. However, isotopic change rate can be variable even between the life stages of a single species, which affects field data interpretation. We conducted diet‐switch experiments to compare the δ13C and δ15N changes in multiple tissues between the growing and reproductive stages of ayu fish (Plecoglossus altivelis altivelis). Isotopic changes in the muscle and mucus tissues were faster in the growing stage than in the reproductive stage. Isotopic change in the muscle tissue during the reproductive stage was caused mainly by whole‐body growth, whereas allometric growth and/or catabolic turnover rather than whole‐body growth accounted more for the isotopic changes in mucus tissue during both stages. Isotopic change was slower in muscle, mucus and ovary tissues, in that order, probably according to the allometric growth and/or catabolic turnover of each tissue. Our investigation showed that the timing of drastic changes differed between the δ13C and δ15N values, as the first report of the isotopic change in the fish ovary tissue, which suggests that the δ13C and δ15N values in the ovary tissue may reflect diets and/or habitats of different time scales. Trophic discrimination factors varied between tissues examined. Our results allowed for accurate applications of multiple‐tissue isotope analysis to our target fish over all stages. Such approaches to any fish species are more accurate when considering the variability of isotopic changes between and within stages.

Effects of Dietary Omega-3 Fatty Acids (DHA/EPA) and Vitamin E on Fatty Acid Profile of Common Carp, Cyprinus carpio Ovaries

The current study aimed to clarify the fatty acids profile in the breeding stock ovaries of common carp (Cyprinus carpio L.) fed on two food additives. The study was carried out in the Shrimp Hatchery which belonging to the Basrah Agricultural Directorate. Three treatment groups were used, the first treatment (T1) represented as control without any food additives, the second treatment (T2) supplemented with 5 g/kg omega-3 fatty acids, and the third treatment (T3) to which vitamin E was added at a concentration of 200 mg/kg. Fishes were randomly distributed in three replicates for each treatment with six of common carp brood stock per replicate. Nine cages were used which placed in earthen pond. Feeding trail lasted from 29 Oct 2018 to 14 Mar 2019. After feeding fish for 82 days, the oil was extracted from gonads by two methods, the first one by Soxhlet apparatus and the second by Accelerated Solvent Extraction (ASE) method. The fatty acid profiles were analyzed using gas chromatograph by GC/MS (Gas Chromatography Mass spectrometry). Results showed that the content of fatty acids in the ovaries of treated fishes in T1, the presence of saturated and unsaturated omega-3 and omega-6 fatty acids, were monounsaturated fatty acid (C19H36:1) scored the highest percentage (52.55%). Fishes in T2 recorded the presence of saturated and unsaturated omega-3 and omega-6 fatty acids were omega-12 (C18H34:12) recorded the highest percentage (50.02%), while T3 fishes showed the presence of saturated and unsaturated fatty acids from the group of omega-6 fatty acids and the unsaturated fatty acid omega-9 (C18H34:9) record the highest percentage (63.24%).In conclusion this study suggested that the content of fatty acids in fish ovaries reflects the composition of fatty acids in the diet.

Reproductive Capacity and Sex-Ratio of Noemachelius multifasciatus Day from Mandal River, India

Present communication deals with the reproductive capacity and sex ratio of a hill stream ornamental fish, Noemacheilus multifasciatus Day from river Mandal of Garhwal region, Uttarakhand, India. A total 151 specimens were collected for this investigation from Mandal River during October 2020 to September 2021. The morphometric measurements were made within 2-3 days of collection which were total length (TL) fish body weight (F.B.W) ovary length and ovary weight. For the assessment of reproductive capacity, anterior, middle and the posterior part of ovary were taken and number of ova in each sample was counted with the help of binocular microscope. The reproductive capacity in terms of absolute fecundity was calculated as F = S. OW/100. Sex ratio was calculated for whole period of study and its signification was tested by Chi-Square test (χ²). The length of fishes ranged from 56mm to 80mm whereas fish body weight varied from 2138mg to 6347 mg.. The lowest reproductive capacity (175 eggs) was observed for the fish length 56mm, and body weight 2138mg. whereas highest reproductive capacity (3476 eggs) was estimated in fish measuring 80 mm length and fish body weight 6347mg. The reproductive capacity was more dependent on the ovary length (r= 0.9894) and fish weight (r = 0.9812), than the fish ovary weight (0.9786) and fish length (0.9248). The average ratio was 1.16 for female: 1.0 for male.

Influence of broodstock diets on growth, fecundity and spawning performance of swamp eel Monopterus albus

Three experimental diets including frozen trash fish (Diet 1), the equal mixture of frozen trash fish and formulated pellets (Diet 2), and formulated pellets (Diet 3) were prepared and fed to the female broodstocks of Monopterus albus for 6 weeks. Swamp eels fed with Diet 3 exhibited lower values of weight gain and specific growth rate (p < 0.05). Plasma concentrations of total protein, total cholesterol and estradiol were higher in fish fed Diet 1. Fish fed with Diet 3 had higher values of crude protein (CP) and ash, together with lower moisture concentration in muscle. Concentrations tendency of CP, ash and moisture in the ovary were completely opposite to that in the muscle. The concentration of total n−3 fatty acids was highest in both the muscle and ovary of fish fed Diet 1. Fertilization of eggs in fish fed Diet 3 was blocked. It suggested that the insufficiency of plasma estradiol, and n−3 fatty acids in the muscle and ovary, together with the impediment of protein mobilization from muscle to ovary, caused the fertilization failure of M. albus fed with formulated feeds. Equal mixture of trash fish and formulated pellets was appropriate for the successful propagation of swamp eels.

Alterations in ultra-structures and elemental composition of ovaries of Labeo rohita (Hamilton-Buchanan) as a pollution indicator in Harike Wetland (Ramsar Site), India

ABSTRACT Teleosts are the largest class of bony fishes and account for more than 90% of fish species. Their reproductive systems are the key feature to adopt gradual evolutionary change under aquatic environmental conditions. Aquatic ecosystems are faced with a serious threat to their proliferation due to the discharge of untreated industrial effluents into them. Here, scanning electron microscopy (SEM) was used to examine the damaged ovaries of the freshwater fish Rohu, Labeo rohita (Hamilton-Buchanan), due to their exposure to different heavy metals in the polluted waters of Harike Wetland in Punjab, India. Deformed oocytes with rough and distorted surfaces were observed in SEM. Microscopic analysis showed that environmental pollution caused by municipal effluents had damaged the microstructural surface on the fish ovaries. Energy-dispersive X-ray spectroscopy also showed damage of the microstructural surface and an abnormal deposition of heavy metals. The alterations in the reproductive system of the fish appeared to be the most responsive and a reliable indicator of environmental pollution, which indicates their significance in aquatic biomonitoring systems. This is the first report in North India showing tissue level markers in biomonitoring programs using the ovary of Labeo rohita as the model system. Microscopic analysis showed that environmental pollution caused by municipal effluents damaged the microstructural surface of the fish ovaries.

Heavy Metal (Ni, Fe) Concentration in Water and Histopathological of Marine Fish in the Obi Island, Indonesia.

This study aims to determine the content of nickel (Ni) and iron (Fe) as well as histopathological analysis of marine fish in Obi Island waters as a bioindicator of pollution. Besides, water quality conditions were carried out in-situ and ex-situ observations. The parameters observed were temperature, brightness, salinity, pH, dissolved oxygen, nitrate, orthophosphate, ammonia, iron (Fe), and nickel (Ni). The results showed the temperature range between 26.48 ℃ to 27.99 ℃ below the quality standard or low temperature. The brightness of the relationship between 12 m and 13 m is under quality standards. The salinity range between 31.01 ppt to 32.13 ppt below the quality standard. The pH range is from 8.6 to 8.7 in high or alkaline conditions. Ammonia range between 0.4 mg / L to 0.7 mg / L exceeds the quality standard. The range of nitrate between 0.009 mg / L to 0.012 mg / L exceeds the quality standard. The range of phosphate between 0.016 mg / L to 0.019 mg / L exceeds the quality standard. The DO range between 3.68 mg / L to 3.77 mg / L lower than the quality standard. The metal range of 0.6 mg / L to 0.9 mg / L exceeds the quality standard. The range of Ni metal between 0.06 mg / L to 0.09 mg / L exceeds the quality standard. Histopathological analysis showed that the liver had a hemorrhage, degeneration of blood vessels, vacuolate degeneration, necrosis, or cell death. The muscles experience edema, degeneration of muscle fibers, atrophy of muscle bundles, vacuolar degeneration of muscle Bundles, hemorrhage, infiltration of lymphocytes, and necrosis. The intestine experience infiltration of lymphocytes, melanomacrophages, and necrosis. While P. tayenus fish ovaries showed necrosis structure oocytes. This research can be a reference for warning of heavy metal pollution in Obi Island waters, binding to the nature of heavy metals that can accumulate in fish tissue.Keywords: Water quality; Heavy Metal; Pollution; Histopathological; Obi Island.

Reproductive development and maturity stage benchmarking in a freshwater teleost ruffe Gymnocephalus cernuus L. in Loch Lomond.

A wide range of classification schemes has been developed to describe reproductive development and stages in teleosts. These have been rationalised for clarity in recent years by Brown-Peterson et al. (Marine and Coastal Fisheries, 2011, 3, 52-70) and the WKASMSF (ICES, Report of the workshop for advancing sexual maturity staging in fish (WKASMSF), 2018) working groups. These have largely benchmarked classification schemes with examples from seawater fishes but have not been routinely applied to freshwater species. In addition, classification schemes developed to assess maturation of fish ovaries are either macroscopic and can be used in the field or histological for laboratory-based studies, and the two have rarely been coupled in the same classification scale or study. The present study reviews published maturation classifications for application to a freshwater teleost, ruffe Gymnocephalus cernuus L., sampled monthly over three annual cycles in Loch Lomond at locations in the extreme north and south ends of the loch. The gonado-somatic index and size frequency distribution of oocytes were used to verify the annual reproductive cycle and spawning of several egg batches. The study showed that a paired modified Percid scale (Treasurer & Holliday, Journal of Fish Biology, 1981, 18, 359-376) and the Rinchard scale (Rinchard & Kestemont, Journal of Fish Biology, 1996, 49, 883-894) gave an improved macroscopic and histological description of maturation compared with and benchmarked against previous ovarian developmental classifications for G. cernuus. The attributes of these various classifications are benchmarked and compared with the Brown-Peterson and WKMATCH scales, and the merits of these new notations are examined.

Association between Heavy Metals and Selected Reproductive Parameters in the Nile Tilapia, Oreochromis niloticus, along River Ruiru, Kiambu County, Kenya

The Nile tilapia, Oreochromis niloticus, is a tropical fish species of commercial importance in both aquaculture and in the wild. It plays a great role in human nutrition and food security. River Ruiru is one of the rivers faced with pollution from nearby industries. It is inhabited by various species of fish such as tilapia and common carp, which, may be harvested by surrounding communities for food. High levels of heavy metals disrupt normal reproductive process in fish. Besides, it has been reported that edible fish contaminated with heavy metals has deleterious effects on the health of humans and other animals that consume them. There is no documented information on the association between heavy metals and the reproductive biology of Oreochromis niloticus in River Ruiru. This study was aimed at assessing the level of heavy metals in fish ovaries and their association with selected reproductive parameters in the reproductive cycle of O. niloticus. Fish samples were collected monthly, for 8 months, from the downstream and upstream sections. Gonadosomatic index (GSI), serum 17β-estradiol (E2) levels and levels of heavy metals lead, cadmium, copper, iron and zinc in ovaries were determined. The levels of the heavy metals were measured using Atomic Absorption Spectrophotometer. The level of E2 was analyzed using Enzyme-Linked Immunosorbent Assay. There was no significant difference in the GSI between the upstream and the downstream sites (t=0.82, p=0.416). Similarly, there was no significant difference in the levels of E2 between the downstream and the upstream sampling sections. In the downstream, the levels of lead and iron in fish ovaries were significantly higher compared to the upstream (lead: t = 3.36, p = 0.002; iron: t = 4.920, p=0.001). The results showed that levels of heavy metals did not associate with the selected reproductive parameters in the Nile tilapia, along River Ruiru. Levels of lead and cadmium were above allowable concentrations for fish consumption when compared to WHO levels. The study recommends that the Ministry of Environment and Natural Resources should put measures in place to stop discharging raw effluents into River Ruiru.

Characterization and Localization of Calb2 in Both the Testis and Ovary of the Japanese Flounder (Paralichthys olivaceus).

Simple SummaryCalretinin (CALB2), which is a Ca2+-binding protein, plays a known pivotal role in the neural system in vertebrates. The role of CALB2 in mammalian gonads has been gradually recognized; however, little information on the function of CALB2 in fish gonads has been reported. Therefore, we firstly identified the calb2 gene in Paralichthys olivaceus (P. olivaceus), and then investigated its tissue distribution and localization in the gonads by real-time PCR, western blotting, and immunohistochemistry. The P. olivaceus calb2 mRNA was relatively highly expressed in both the testis and ovary. The CALB2 protein is located in Leydig cells of the testis and ovarian germ epithelial cells in P. olivaceus. This study provides a basis for further explorations on the function and regulation mechanism of calb2 in fish gonads.Although its function in mammalian gonads has been gradually recognized, the expression and function of calretinin (CALB2)—a Ca2+-binding protein—in the testis and ovary of fish are still unclear. Here, we identified the cDNA sequences of calb2 in Paralichthys olivaceus (P. olivaceus); analyzed its gene structure and phylogenetic and syntenic relationship by bioinformatics; and investigated its tissue distribution and localization in the gonads by real-time PCR, western blotting, and immunohistochemistry. The P. olivaceus calb2 gene has 11 exons and 10 introns, and the full-length cDNA is 1457 bp, including an open reading frame (ORF) of 816 bp encoding 271 amino acids. The CALB2 of P. olivaceus has a higher homology with Lates calcarifer (99%) compared with other species. The conserved synteny of calb2 neighboring gene loci was also detected in fish. Real-time PCR showed that the expression of calb2 mRNA is abundant not only in the brain, but also in the gonads, and exhibits a higher expression in the testis than in the ovary. Western blotting indicated that the CALB2 protein has a higher expression in the testis compared with the ovary. Immunohistochemistry demonstrated that the CALB2 protein appears in Leydig cells and the ovarian germ epithelium. These results reveal that calb2 plays an important role in the gonads of P. olivaceus.

Food availability alters expression profiles of genes in relation to reproduction and nutrition in the females of tropical damselfish (Chrysiptera cyanea).

This study evaluated the effects of food availability on the transcript levels of genes related to reproduction and growth in the sapphire devil (Chrysiptera cyanea), a tropical damselfish. Nonbreeding fish were reared at high-food (HF) and low-food (LF) levels for 4 weeks under long-days. Vitellogenic oocytes could be observed in the ovaries of the HF group. The quantitative polymerase chain reaction analysis revealed that lhβ and cyp19b in the brains, vtg and igf1 in the livers and cyp19a in the ovaries of HF fish were significantly higher than that of LF fish, suggesting that estradiol-17β (E2) synthesis in the ovary and brain is activated when suitable permissive factors are available to fish. Food limitation lowered hepatic igf1 and dio2, suggesting that the TH-IGF1 signaling system functions in the liver, and that food availability altered hepatic deiodination activities related to intercellular levels of thyroid hormones. Hepatic dio2 significantly decreased when fish were immersed for 3 days in E2-containing seawater; this suggests that E2 impedes the conversion of T4 to T3 in the liver. Our study shows that igf1 was upregulated in accordance with HF-induced vitellogenesis but downregulated by E2 treatment, suggesting that igf1 is bidirectional and altered by maturational status. Once vitellogenesis begins under a suitable range of proximal factors, fish need to maintain their nutritional status because food availability is a permissive factor for their reproduction.

C-ECi: a CUBIC-ECi combined clearing method for three-dimensional follicular content analysis in the fish ovary†.

Deciphering mechanisms of oocyte development in the fish ovary still remain challenging, and a comprehensive overview of this process at the level of the organ is still needed. The recent development of optical tissue clearing methods has tremendously boosted the three-dimensional (3D) imaging of large size biological samples that are naturally opaque. However, no attempt of clearing on fish ovary that accumulates extremely high concentration of lipids within oocytes has been reported to date. To face with this ovarian-specific challenge, we combined two existing clearing methods, the nontoxic solvent-based ethyl cinnamate (ECi) method for efficient clearing and the Clear Unobstructed Brain Imaging Cocktails and Computational (CUBIC) method to enhance lipid removal and reduce nonspecific staining. The methyl green fluorescent dye was used to stain nuclei and delineate the follicular structures that include oocytes. Using this procedure (named CUBIC-ECi [C-ECi]), ovaries of both medaka and trout could be imaged in 3D and follicles analyzed. To our knowledge, this is the first procedure elaborated for clearing and imaging fish ovary in 3D. The C-ECi method thus provides an interesting tool for getting precise quantitative data on follicular content in fish ovary and promises to be useful for further developmental and morphological studies.

Screening of potential uranium protein targets in fish ovaries after chronic waterborne exposure: Differences and similarities between roach and zebrafish

Concentration of uranium (U), a naturally encountered radioactive element in earth's crust, can be enhanced in freshwater ecosystems (μg.L−1 – mg.L−1) due to various anthropogenic activities. The consequent aquatic organism exposure to U leads to its accumulation in all organs, particularly in the gonad, and in subcellular fractions (mainly the cytosol); then it is known to affect fish at several biological levels, and more particularly, at a reproduction endpoint, with a decrease in the total number of eggs, spawn events and larvae survival. The understanding of U reprotoxicity requires the fine knowledge of its speciation at molecular level, i.e., its interaction with cytosolic biomolecules. In this study, we focus on the U-protein interactions in gonads. A non-denaturating extraction protocol combined with size exclusion chromatography (SEC) allowed the separation of metal-protein complexes in ovaries of U-contaminated wild roaches before their elemental detection (ICP MS). This enables unprecedented information to be obtained about U distribution in ovaries of autochthonous fish, Rutilus rutilus, which is different in some points from that obtained in the model species, Danio rerio under controlled laboratory conditions at a similar concentration level. Finally, the ability to transpose results from model to autochthonous fish was briefly discussed.

Transcriptional Changes in the Ovaries of Perch from Chernobyl.

Fish have been highly exposed to radiation in freshwater systems after the Chernobyl Nuclear Power Plant (NPP) accident in 1986 and in freshwater and marine systems after the more recent Fukushima NPP accident in 2011. In the years after the accident, the radioactivity levels rapidly declined due to radioactive decay and environmental processes, but chronic lower dose exposures persisted. To gain insights into the long-term effects of environmental low dose radiation on fish ovaries development, a high-throughput transcriptomic approach including a de novo assembly was applied to different gonad phenotypes of female perch: developed gonads from reference lakes, developed/irradiated from medium contaminated lake, and both developed/irradiated and undeveloped from more highly contaminated lakes. This is the most comprehensive analysis to date of the gene responses in wildlife reproductive system to radiation. Some gene responses that were modulated in irradiated gonads were found to be involved in biological processes including cell differentiation and proliferation (ggnb2, mod5, rergl), cytoskeleton organization (k1C18, mtpn), gonad development (nell2, tcp4), lipid metabolism (ldah, at11b, nltp), reproduction (cyb5, cyp17A, ovos), DNA damage repair (wdhd1, rad51, hus1), and epigenetic mechanisms (dmap1). Identification of these genes provides a better understanding of the underlying molecular mechanisms underpinning the development of the gonad phenotypes of wild perch and how fish may respond to chronic exposure to radiation in their natural environment, though causal attribution of gene responses remains unclear in the undeveloped gonads.

Growth differentiation factor 9 (gdf9) and bone morphogenetic protein 15 (bmp15) are potential intraovarian regulators of steroidogenesis in Japanese flounder (Paralichthys olivaceus)

Members of transforming growth factor-β (TGF-β) superfamily are vital regulators during the development of fish ovary. However, its intraovarian functions in teleost are still unclear. As members of the TGF-β superfamily, gdf9 and bmp15 are necessary for follicle formation and granulosa cell proliferation. Here in Japanese flounder, quantitative real-time polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH) analysis showed that gdf9 and bmp15 were mainly expressed in oogonia and oocytes, whereas weakly expressed in non-ovarian tissues. Overexpression of single gdf9 and the co-overexpression with bmp15 could up-regulate the expression of most steroidogenic genes, while the overexpression of single bmp15 could down-regulate the expression of most steroidogenic genes. These findings demonstrate that single gdf9 and the combination with bmp15 may act as “activator”, while single bmp15 may act as “inhibitor” in the process of steroidogenesis in flounder. This was also verified in negative feedback regulation of gdf9 and bmp15 during hormone treatment. High concentration of human chorionic gonadotropin (hCG) could down-regulate gdf9 and up-regulate bmp15, which were beneficial for the homeostasis of hCG hormone. Besides, knockdown of either gdf9 or bmp15 could significantly down-regulate most steroidogenic genes. This indicated that heterodimer of GDF9:BMP15 might be the most bioactive ligand in gonad development of flounder. Taken together, our study provided a novel recognition that gdf9 and bmp15 could regulate steroidogenesis in teleost through mechanism different from that in mammals.

Expression profile and estrogenic regulation of Amh during gonadal sex differentiation in northern snakehead (Channa argus).

Anti-Müllerian hormone (Amh) plays a critical role in both early sex determination and later gonad development in vertebrate species. However, it remains unknown in northern snakehead (Channa argus), which is economically important freshwater fish with sexual dimorphism. This study aimed to identify the expression profiles and estrogenic regulation of CaAmh during gonadal sex differentiation in C. argus. The cDNA and genomic DNA sequences of CaAmh were identified by PCR and RACE techniques. The expression patterns of CaAmh were detected by qRT-PCR during the gonadal sex differentiation and after 17α-ethinyloestradiol (EE2) treatments. CaAmh is composed of seven exons and six introns, and its full-length cDNA is 2413 bp in length, with 1635 bp open reading frame (ORF) that encodes a 544 amino acid protein. Tissues expression patterns revealed that CaAmh display the highest expression in testis of XY males (40.36 folds, p < 0.01). The spatio-temporal expression patterns during gonadal sex differentiation indicated that CaAmh expression differed between XX females and XY males at 30 day after hatching (dah), and reached to the peak (36.03 folds, p < 0.01) at 90 dah in XY gonads. However, CaAmh expression in XX gonads remained low throughout the sampling period. Furthermore, CaAmh expression in the gonads (ovaries) of the sex-reversed XY fish (XY-F) by the administration of estrogen EE2 was downregulated to low level, similar to that in ovaries of normal XX females (XX-F). These results show that Amh plays a critical role in testicular differentiation of C. argus and it is apparently modulated by estrogens in this species.