Plasma Vitellogenin Research Articles

A Quantitative Adverse Outcome Pathway for Embryonic Activation of the Aryl Hydrocarbon Receptor of Fishes by Polycyclic Aromatic Hydrocarbons Leading to Decreased Fecundity at Adulthood.

Quantitative adverse outcome pathways (qAOPs) describe the response-response relationships that link the magnitude and/or duration of chemical interaction with a specific molecular target to the probability and/or severity of the resulting apical-level toxicity of regulatory relevance. The present study developed the first qAOP for latent toxicities showing that early life exposure adversely affects health at adulthood. Specifically, a qAOP for embryonic activation of the aryl hydrocarbon receptor 2 (AHR2) of fishes by polycyclic aromatic hydrocarbons (PAHs) leading to decreased fecundity of females at adulthood was developed by building on existing qAOPs for (1) activation of the AHR leading to early life mortality in birds and fishes, and (2) inhibition of cytochrome P450 aromatase activity leading to decreased fecundity in fishes. Using zebrafish (Danio rerio) as a model species and benzo[a]pyrene as a model PAH, three linked quantitative relationships were developed: (1) plasma estrogen in adult females as a function of embryonic exposure, (2) plasma vitellogenin in adult females as a function of plasma estrogen, and (3) fecundity of adult females as a function of plasma vitellogenin. A fourth quantitative relationship was developed for early life mortality as a function of sensitivity to activation of the AHR2 in a standardized in vitro AHR transactivation assay to integrate toxic equivalence calculations that would allow prediction of effects of exposure to untested PAHs. The accuracy of the predictions from the resulting qAOP were evaluated using experimental data from zebrafish exposed as embryos to another PAH, benzo[k]fluoranthene. The qAOP developed in the present study demonstrates the potential of the AOP framework in enabling consideration of latent toxicities in quantitative ecological risk assessments and regulatory decision-making. Environ Toxicol Chem 2024;43:2145-2156. © 2024 SETAC.

Dietary carbohydrate to lipid ratio affects growth, reproductive performance and health of female yellow catfish (Pelteobagrus fulvidragrus): a lipidomics analysis

This study aimed to examine the impact of dietary carbohydrate to lipid (CHO/L) ratio on the growth, reproductive, and offspring performance of broodstock yellow catfish, and to elucidate the metabolic differences between mothers and offspring using lipidomics. Five isonitrogenous and isoenergetic diets with varying CHO/L ratios (0.65, 1.44, 2.11, 3.13, and 5.36) were fed to three groups of 35 female broodfish in a pond-cage culture system. After an eight-week feeding trial, the dietary CHO/L ratio had a significant impact on the growth and reproductive performance of female yellow catfish. The weight gain ratio (WGR) and specific growth rate (SGR) in the CHO/L0.65 and CHO/L2.11 groups were significantly higher than those in the CHO/L5.36 group (P < 0.05). The fertilization and hatching rates were the highest when the dietary CHO/L ratio was 0.65 and 2.11, respectively. When the dietary CHO/L ratio was 3.13 and 5.36, the plasma contents of testosterone (T) was significantly lower than those of other groups (P = 0.013), and the plasma vitellogenin (VTG) content was the lowest when the CHO/L ratio was 5.36. The plasma contents of estradiol (E2) significantly decreased with increasing dietary CHO/L ratio (PL = 0.012). Lipidomic analysis revealed that the ovary primarily consisted of five subclasses in terms of lipid composition, namely triglyceride, fatty acyl, sterol, glycerophospholipid, and sphingolipid; however, sphingolipids were not detected in the larvae. The relative expression levels of the ovarian lipid metabolism-related genes sterol regulatory element binding protein 1 (srebp1), acetyl-coa carboxylase (acc), delta (12)-oleate desaturase (fad2), and elongation of very long chain fatty acids protein 5 (elvol5) significantly increased with increasing dietary CHO/L ratio (P < 0.05). The relative expression levels of lipid metabolism-related genes srebp 1, peroxisome proliferator activated receptor α (pparα), carnitine palmitoyl transferase 1 isoform (cpt), adipose triglyceride lipase (atgl), fad2, and elvol5 in offspring larvae were initially increased and then decreased with increasing dietary CHO/L ratios until reaching a maximum at a ratio of 2.11 (P < 0.05). In conclusion, based on the broken-line regression of the dietary CHO/L ratio and egg diameter, the optimal dietary CHO/L ratio was 1.91 for broodfish yellow catfish. A high-CHO/L ratio diet results in increased lipogenesis and hepatic lipid accumulation in maternal organisms, leading to impaired reproductive performance and reduced offspring quality.

Intraperitoneal administration of kisspeptin-10 modulates follicle maturation, gonadal steroids, calcium and metabolites in Sterlet sturgeon, Acipenser ruthenus

Kisspeptin is a multifunctional neurohormone, primarily involved in the regulation of reproduction. We tested whether peripheral administration of kisspeptin10 (KP-10) via intraperitoneal injection or slow release affects reproductive hormones and metabolites in Sterlet sturgeon (Acipenser ruthenus). Plasma and mucus 17β-estradiol (E2), and testosterone (T), plasma and follicular vitellogenin (VTG) and calcium (Ca) as well as glucose and lipids were determined. Mature Sterlet sturgeon were grouped into six groups: saline i.p injection (control), human kisspeptin (hKP-10) i.p injection; acipenser kisspeptin (aKP-10) i.p injection; hKP-10 (slow release); aKP-10 (slow-release) and no treatment control. No effect for KP-10 on sturgeon body weight was found after 4 weeks of treatment. Multivariate analysis revealed a significant disparity in plasma E2 levels. It was significantly different between groups (time, P = 0.0022). E2 in epithelia mucosa showed significant difference between and within groups in the acute group (time, P = 0.0252; treatment, P = 0.0423; time × treatment, P = 0.0429). T levels were unaffected by treatments (P > 0.05). The presence of synthetic aKP-10 led to an elevation in oocyte and plasma VTG levels (P < 0.05). Prolonged exposure to this peptide resulted in an increase in plasma calcium levels. Simultaneously, there was an augmentation in the number of mature follicles. Regardless of the duration of exposure, aKP-10 significantly elevated plasma glucose levels in Sterlet (P < 0.0). Additionally, KP-10 led to an increase in plasma lipids and cholesterol in Sterlet. Overall, our data support an involvement for KP-10 in the regulation of gonadal steroid hormones, oocyte maturation and metabolite levels in sturgeon, suggesting a positive role for this peptide in the reproductive physiology of this species.

Higher intake of β-glucan impairs reproduction in a female teleost, Tor putitora (Hamilton, 1822).

Although the immuno-modulatory and stress-relieving properties of β-glucan is well elucidated in humans and other animal models, including fish, its role as a dietary supplement on reproduction is extremely scarce. Therefore, in this study, adult female fish were fed one of four test diets having 0 (control), 0.5, 1, and 1.5% β-D-glucan for 130 days and its effect on reproductive performance, ovarian and liver histology, sex hormones, and transcript abundance of selected reproduction-related genes was assessed. Low dietary intake of β-glucan improved fertilization and hatching rates (p<0.05). The relative fecundity and percentage of spawning females were higher (non-significant) in 0.5% β-glucan-fed groups. Surprisingly, even after 130 days, spawning did not occur in 1.5% β-glucan-fed individuals. Irrespective of β-glucan intake, all the brooders recorded similar plasma 17β-estradiol and maturation-inducing hormone (p>0.05). Higher intake of β-glucan (1.5%) upregulated aromatase genes without a parallel increase in 17β-estradiol. However, plasma vitellogenin increased with increasing β-glucan up to 1.0% then declined at 1.5% (p<0.05). The fish that received control, 0.5, and 1.5% β-glucan recorded similar vitellogenin levels in their plasma. Significantly higher plasma cortisol was evidenced in 1.5% β-glucan fed brooders (p<0.05). Histologically, higher follicular atresia and leaking of yolk material was evidenced in 1.5% β-glucan-fed group. Liver histology revealed the highest nutrient/lipid accumulation in fish that received 1.0% and 1.5% β-glucan. This study demonstrated the stimulatory effect of β-glucan intake at a lower dose (0.5%) on reproduction. However, higher intake (1.5%) could perturb normal reproductive function in a fish model and caused an increased number of atretic follicles leading to spawning/reproductive failure.

Endocrine patterns associated with ovarian development in female Pacific halibut (Hippoglossus stenolepis)

The Pacific halibut (Hippoglossus stenolepis) is a large migratory demersal flatfish species that occupies a top trophic role in the North Pacific Ocean and Bering Sea ecosystems, where it also supports various fisheries. As a first attempt to characterize the endocrine mechanisms driving sexual maturation in this important species, we collected pituitary, ovarian and blood samples from Pacific halibut females captured in the wild that were classified histologically into various female developmental stages. We conducted gene expression analyses of gonadotropin beta subunits in the pituitary and observed that mRNA expression levels of fshb gradually increased throughout vitellogenesis, remained elevated until before ovulation and declined after spawning. In contrast, the mRNA expression levels of lhb markedly increased during oocyte maturation and remained elevated until after spawning. Ovarian mRNA expression levels of the gonadotropin receptor genes fshr and lhr peaked during oocyte maturation and before spawning, respectively, immediately following the developmental stage at which pituitary fshb and lhb mRNA expression first reached maximum levels. The ovarian gene expression patterns of steroidogenic enzyme genes cyp19a1 and hsd20b2 paralleled those of fshr and lhr, respectively. Testosterone and 17β-estradiol (E2) plasma levels increased concomitantly with fshr and cyp19a1 mRNA expression levels, and vitellogenin plasma levels increased throughout vitellogenesis and reached maximum levels prior to spawning. These results are consistent with the notion that in female Pacific halibut, as in other teleosts, vitellogenesis and oocyte maturation and ovulation are likely under the control of pituitary gonadotropic hormones Fsh and Lh, respectively.

Endocrine Disruption Is Reduced but Still Widespread in Wild Roach (Rutilus rutilus) Living in English Rivers.

Endocrine disruption of wild fish, primarily resulting in the feminization of males, has been reported in English river sites for several decades. Estrogenic activity emanating from wastewater treatment works (WwTW) has been conclusively demonstrated to be the main driver of these feminized phenotypes. Here, we revisit 10 English river sites previously surveyed in the late 1990s and early 2000s to assess how the frequency and severity of feminization now compare with the historical surveys. In the contemporary assessment, 60% of the sites revisited still showed endocrine disruption at the tissue organization level (oocytes present in otherwise male gonads; intersex) and 90% of sites had average male plasma vitellogenin concentrations (female-specific yolk protein; a sensitive biomarker of estrogen exposure) above natural baseline levels. In contrast to the historic surveys, none of the males sampled in the contemporary survey had ovarian cavities. At one of the larger WwTW, improvements to treatment technology may have driven a significant reduction in intersex induction, whereas at several of the smaller WwTW sites, the frequencies of feminization did not differ from those observed in the late 1990s. In conclusion, we show that although the severity of feminization is now reduced at many of the revisited sites, endocrine-disrupting chemicals are still impacting wild fish living downstream of WwTW in England.

Putative adverse outcome pathway development based on physiological responses of female fathead minnows to model estrogen versus androgen receptor agonists

Several adverse outcome pathways (AOPs) have linked molecular initiating events like aromatase inhibition, androgen receptor (AR) agonism, and estrogen receptor (ER) antagonism to reproductive impairment in adult fish. Estrogen receptor agonists can also cause adverse reproductive effects, however, the early key events (KEs) in an AOP leading to this are mostly unknown. The primary aim of this study was to develop hypotheses regarding the potential mechanisms through which exposure to ER agonists might lead to reproductive impairment in female fish. Mature fathead minnows were exposed to 1 or 10 ng 17α-ethynylestradiol (EE2)/L or 10 or 100 µg bisphenol A (BPA)/L for 14 d. The response to EE2 and BPA was contrasted with the effects of 500 ng/L of 17β-trenbolone (TRB), an AR agonist, as well as TRB combined with the low and high concentrations of EE2 or BPA tested individually. Exposure to 10 ng EE2/L, 100 µg BPA/L, TRB, or the various mixtures with TRB caused significant decreases in plasma concentrations of 17β-estradiol. Exposure to TRB alone caused a significant reduction in plasma vitellogenin (VTG), but VTG was unaffected or even increased in females exposed to EE2 or BPA alone or, in most cases, in mixtures with TRB. Over the course of the 14-d exposure, the only treatments that clearly did not affect egg production were 1 ng EE2/L and 10 µg BPA/L. Based on these results and knowledge of hypothalamic–pituitary–gonadal axis function, we hypothesize an AOP whereby decreased production of maturation-inducing steroid leading to impaired oocyte maturation and ovulation, possibly due to negative feedback or direct inhibitory effects of membrane ER activation, could be responsible for causing adverse reproductive impacts in female fish exposed to ER agonists.

Using Plasma Vitellogenin in Loggerhead Sea Turtles to Assess Reproductive Maturation and Estrogen-Like Contaminant Exposure.

Vitellogenin (VTG), an egg yolk precursor, is abnormally produced by male and juvenile oviparous species after exposure to estrogens. Plasma VTG in loggerhead sea turtles (Caretta caretta) helped us understand their reproductive maturation and investigate it as a biomarker of contaminant exposure. The presence of VTG was screened in plasma from 404 loggerheads from the northwestern Atlantic Ocean using a freshwater turtle antibody in western blots. The concentrations of VTG were semiquantified using band intensities calibrated to results from a loggerhead antibody enzyme-linked immunoassay. The detection and concentrations of VTG were in (from highest to lowest): nesting females, in-water adult females, subadult females, smaller females, unknown sex, and males. Loggerheads from this region begin vitellogenesis at ≅77 cm straight carapace length. We classified VTG expression as abnormal in nine male or juvenile turtles. Organochlorine contaminant (OC) concentrations were measured in blood and/or fat biopsies of some turtles. One abnormal VTG female had the second highest fat polychlorinated biphenyl (PCB) and 4,4'-dichlorodiphenyldichloroethylene concentrations compared among 43 VTG-negative juveniles. The nine VTG-abnormal turtles had average blood PCB concentrations 8.5% higher, but not significantly different, than 46 VTG-negative juveniles (p = 0.453). In turtles less than 77 cm, blood PCB concentrations were significantly, but weakly, correlated with semiquantified VTG concentrations (tau = 0.1, p = 0.004). Greater blood OC concentrations were found in adult females than in males, which motivated the creation of a conceptual model of OC, VTG, and hormone concentrations across a reproductive cycle. A decision tree is also provided incorporating VTG as a sexing tool. Abnormal VTG expression cannot conclusively be linked to endocrine disruption caused by these OC concentrations. Studies should further investigate causes of abnormal VTG expression in wild sea turtles. Environ Toxicol Chem 2023;42:1309-1325. © 2023 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

A multi-level assessment of biological effects associated with mercury concentrations in smallmouth bass, Micropterus dolomieu

Total mercury (THg) was measured in muscle (fillet) and liver tissue of adult smallmouth bass Micropterus dolomieu collected at multiple sites in the Potomac and Susquehanna River drainages within the Chesapeake Bay watershed. Smallmouth bass in these drainages have experienced episodic mortality events, a high prevalence of skin lesions and reproductive endocrine disruption (intersex or testicular oocytes and plasma vitellogenin in males). A multi-level assessment of general and reproductive health including indicators at the organismal, organ, cellular and molecular levels was conducted on adult smallmouth bass during the spring (prespawn) season. Concentrations of THg were correlated with increased visible abnormalities, increased macrophage aggregates and tissue parasite burdens. In male bass positive correlations of THg were observed with plasma vitellogenin and hepatic transcript abundance of estrogen receptor β1 and androgen receptor α, while there was a negative association with estrogen receptors α and β2 and androgen receptors β. In female bass there was a negative correlation between THg and plasma vitellogenin as well as hepatic transcript abundance of vitellogenin, choriogenin, estrogen receptor β2 and 17β hydroxysteroid dehydrogenase. Associations of THg concentrations with various biological indicators suggest mercury may be an important environmental stressor contributing to the observed adverse effects in smallmouth bass populations.

Plasma Vitellogenin and Testosterone in Diamond-backed Terrapins (Malaclemys terrapin) during the Nesting Season in Coastal New Jersey

Vitellogenesis is the process in which female oviparous vertebrates synthesize the protein vitellogenin to develop egg yolk. In some species, vitellogenin has been used to investigate reproductive status, as a biomarker of clutch size and nesting frequency, and as a biomarker of exposure to endocrine-disruptive chemicals. The Diamond-backed Terrapin (Malaclemys terrapin) is an obligate coastal species that in the northern extent of its range nests in the spring and summer months. Diamond-backed Terrapins serve as a key indicator species of coastal ecosystem health; thus, furthering our understanding of the endocrine control of reproduction may inform biologists of the health and status of coastal ecosystems. The objective of this study was to quantify baseline values of vitellogenin and testosterone in Diamond-backed Terrapins during the nesting season, in the northern part of their range. Blood samples were taken from adult female terrapins from two populations in coastal New Jersey from June–August. Enzyme-linked immunosorbent assays (ELISAs) were used to quantify vitellogenin (VTG) and testosterone (T) across the nesting season. VTG concentrations showed peak values in the earliest part of the nesting season, significantly declining through the summer before reaching basal values in August, with T showing a similar trend. This suggests that terrapins in New Jersey follow a similar reproductive cycle to other turtle species from temperate latitudes. Additionally, we found that larger females exhibited higher concentration of T and VTG than smaller females. This suggests that VTG and T are useful biomarkers of reproductive output in these animals. Lastly, we also noted that larger females tended to nest earlier in the nesting season than smaller females. We hypothesize that larger females may compete for resources more effectively and efficiently than smaller females, which may confer larger individuals a fitness advantage.

A case study: temporal trends of environmental stressors and reproductive health of smallmouth bass (Micropterus dolomieu) from a site in the Potomac River Watershed, Maryland, USA

Decades of poor reproductive success and young-of-the-year survival, combined with adult mortality events, have led to a decline in the smallmouth bass (SMB; Micropterus dolomieu) population in sections of the Potomac River. Previous studies have identified numerous biologic and environmental stressors associated with negative effects on SMB health. To better understand the impact of these stressors, this study was conducted at the confluence of Antietam Creek and the Potomac River from 2013 to 2019 to identify temporal changes associated with SMB reproductive health. Surface water samples were collected and analyzed for over 300 organic contaminants, including pesticides, phytoestrogens, pharmaceuticals, hormones and total estrogenicity (E2Eq). Adult SMB were collected and sampled for multiple endpoints, including gene transcripts associated with reproduction (molecular), histopathology (cellular), and organosomatic indices (tissue). In males, biomarkers of estrogenic endocrine disruption, including testicular oocytes (TO) and plasma vitellogenin (Vtg) were assessed. Numerous agriculture-related contaminants or land use patterns were associated with gene transcript abundance in both male and female SMB. Positive associations between pesticides in the immediate catchment with TO severity and E2Eq with plasma Vtg in males were identified. In males, the prevalence of TO and detectable levels of plasma Vtg, liver vitellogenin transcripts (vtg) and testis vtg were high throughout the study. Peaks of complex mixtures of numerous contaminants occurred during the spring/early summer when spawning and early development occurs and to a lesser extent in fall/winter during recrudescence. Management practices to reduce exposure during these critical and sensitive periods may enhance reproductive health of these economically important sportfishes.

Immuno-Enzymatic and Proteomic Approaches for Sexing the African Bonytongue (Heterotis niloticus Cuvier, 1829)

Heterotis niloticus is an African species of Osteoglossiformes that presents biological peculiarities and zootechnical performances favorable for fish farming. However, the absence of a sexual dimorphism hinders the optimization of its reproduction in captivity and limits the understanding of its reproductive behavior. This study is aimed at developing a minimally invasive and reliable sexing method to detect vitellogenin (Vtg) in female plasma. A commercial sexing kit (Acobium, Montpellier, France) for Arapaima gigas—a phylogenetically sister species of H. niloticus—successfully identified only 20% of mature H. niloticus females. Enzyme-linked immunosorbent assays (ELISA) were carried out using three Vtg antibodies. The A. gigas Vtg1 antibody cross-reacted significantly with plasma dilutions of female H. niloticus ranging from 1:1000 to 1:10,000, but with relatively low intensity. The Vtg antibody from Osteoglossum bicirrhosum, another species of Osteoglossiformes, showed non-specific binding with the Vtg of H. niloticus female plasma. Finally, an antibody for H. niloticus Vtg developed in this study allowed us to differentiate the two sexes with plasma coating dilutions ranging from 1:1000 to 1:10,000. The results of the assay were validated by a proteomic approach showing that Vtg-targeted mass spectrometry analysis of H. niloticus blood protein extracts could be used to accurately determine the presence of Vtg in the plasma of mature females. The final validation of the ELISA technique using the H. niloticus Vtg antibody was confirmed by visual sexing of a significant number of blood-sampled fish gonads; 100% of the fish were correctly sexed by the ELISA method.

Integrated estrogenic effects and semi-volatile organic pollutants profile in secondary and tertiary wastewater treatment effluents in North China

Endocrine-disrupting effects on aquatic organisms caused by wastewater discharging have raised extensive concerns. However, the efficiency of various wastewater treatment processes to remove estrogenic activity in effluents and the association with organic micropollutants was not well known. We evaluated the estrogenic activity using a well-characterized in vivo bioassay featuring the Chinese rare minnows (Gobiocypris rarus) and analyzed 886 semi-volatile organic compounds (SVOCs) in effluents from four secondary wastewater treatment plants (SWTP A-D) and a tertiary wastewater treatment plant (TTP E) that utilized various common treatment processes in northern China. The final effluents from SWTPs and TTP E all exhibited estrogenic effects, increasing male fish plasma vitellogenin (VTG) contents and estradiol/testosterone (E2/T) ratios. Key regulating genes in the male fish liver including vtg1, vtg3, era, erβ, and cyp19a were upregulated. TTP E demonstrated high performance in reducing estrogenic activity in the effluents, with a reduction of 64% in integrative biomarkers of estrogenic response (IBR). UV disinfection at SWTPs removed IBR by 14%− 33%, while ozone disinfection at TTP E did not reduce IBR. Several SVOCs including alkanes, chlorobenzenes, and phthalates, detected at ng/L to µg/L level, significantly correlated with effluent estrogenic activity. Our findings suggest the necessity and the potential means to improve the efficiency of current wastewater treatment approaches to achieve better protection for aquatic organisms against the joint effects of mixtures of various categories of micropollutants in effluents.

Long term exposure to tris (2-chloroethyl) phosphate (TCEP) causes alterations in reproductive hormones, vitellogenin, antioxidant enzymes, and histology of gonads in zebrafish (Danio rerio): In vivo and computational analysis

In aquatic milieus, tris (2-chloroethyl) phosphate (TCEP) was detected as an emerging environmental contaminant. In this study, in vivo experiment and in-silico docking was integrated systematically to explore the toxic mechanisms of TCEP using zebrafish (Danio rerio). Fish (mean weight of 0.24 ± 0.02 g) were exposed to 100 and 1500 μg L−1 concentrations of TCEP for 28 days under the static renewal method. During chronic exposure, plasma steroid hormones such as testosterone (T) and 17β estradiol (E2), plasma vitellogenin (Vtg) and antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and lipid peroxidation (LPO) in gonads were significantly (P < 0.05) altered in TCEP exposed group (1500 μg L−1) compared to the control group. However, the alterations of these parameters were not significant on the 14th day (except Vtg and GR in testis) in 100 μg L−1 of TCEP exposed groups. There were no significant differences (p > 0.05) in the growth parameters comparing TCEP exposed groups with the control group. The gonads of fish exposed to TCEP showed significant histopathological changes when compared to the control groups. A docking study observed that TCEP possessed binding affinity with the estrogen receptor (ERβ) and androgen receptor (AR). These data indicate that TCEP at tested concentrations adversely affects the aquatic organisms.

Effect of the concentration of estradiol and vitellogenin in the blood serum of meat and egg breed chickens on egg productivity

The aim of the research is to determine the effect of changes in the concentration of estradiol-17β on the concentration of vitellogenin at the stage of egg shell formation and preparation of the body of meat and egg breed chickens (for example, the Pushkin breed) for ovulation.Materials and methods. The material for the study was the Pushkin breed chickens, in the amount of 28 heads. at the age of 48 weeks. Chickens were kept in individual cages. Light mode - 14C:10T. Drinking - nipple, temperature in the house and feeding - according to the standards adopted in the bioresource collection of RRIFAGB "Genetic collection of rare and endangered chicken breeds." Within 10 days prior to blood sampling, an ultrasound scan of the ovary and oviduct was performed from the studied group of chickens, which made it possible to determine the stage of egg formation. Blood from the studied chickens was taken in the morning on an empty stomach for the simultaneous determination of the concentration of estradiol and vitellogenin at the stage of egg shell formation. The concentration of estradiol and vitellogenin in the blood plasma of chickens was determined by ELISA. To clarify the effect of the concentration of estradiol (E2) on vitellogenin (VTG), the studied flock was divided into 2 groups according to the concentration of vitellogenin: 1st gr. – &gt; Mav. (&gt; 20 μg / ml) and 2nd gr. – &lt; Mav. (&lt;20 μg / ml). During the study period, eggs of chickens were evaluated according to the following morphometric parameters: the weight of the egg, yolk, protein and shell, elastic deformation, shape index, density of protein fractions, thickness of the shell and subshell, marbling. The number of eggs laid was determined in 10-day trial period.Results. It has been established that the duration of the ovulatory cycle in Pushkin breed chickens is 27-29 hours. As a result of such a long ovulatory cycle in the following days, laying of eggs occurs later with a progressive shift, as a result of which the laying cycle on average for the studied group of chickens is 3 days. The E2 level in the 1st group was 258,48 ± 30,60 pg/ml (p &lt; 0,05), which significantly differed from the 2nd group – 181,45 ± 13,81 pg/ml. A positive correlation was found between VTG and E2, r = 0,5 (p &lt; 0,001).Conclusion. The VTG level significantly increases in Pushkin chickens at the age of 48 weeks with an increase in the concentration of E2 in the blood. A positive correlation was noted between these features. According to the results of our research, the high content of estradiol and vitellogenin in the chickens blood serum did not have a significant effect on egg productivity.

Reproductive health and endocrine disruption in smallmouth bass (Micropterus dolomieu) from the Lake Erie drainage, Pennsylvania, USA

Smallmouth bass Micropterus dolomieu were sampled from three sites within the Lake Erie drainage (Elk Creek, Twentymile Creek, and Misery Bay, an embayment in Presque Isle Bay). Plasma, tissues for histopathological analyses, and liver and testes preserved in RNALater® were sampled from 30 smallmouth bass (of both sexes) at each site. Liver and testes samples were analyzed for transcript abundance with Nanostring nCounter® technology. Evidence of estrogenic endocrine disruption was assessed by the presence and severity of intersex (testicular oocytes; TO) and concentrations of plasma vitellogenin in male fish. Abundance of 17 liver transcripts associated with reproductive function, endocrine activity, and contaminant detoxification pathways and 40 testes transcripts associated with male and female reproductive function, germ cell development, and steroid biosynthesis were also measured. Males with a high rate of TO (87–100%) and plasma vitellogenin were noted at all sites; however, TO severity was greatest at the site with the highest agricultural land cover. Numerous transcripts were differentially regulated among the sites and patterns of transcript abundance were used to better understand potential risk factors for estrogenic endocrine disruption. The results of this study suggest endocrine disruption is prevalent in this region and further research would benefit to identify the types of contaminants that may be associated with the observed biological effects.

Seasonal Changes in Plasma Hormones, Sex-Related Genes Transcription in Brain, Liver and Ovary during Gonadal Development in Female Rainbow Trout (Oncorhynchus mykiss)

The purpose of this study was to investigate the periodic seasonal changes in endocrine activity and gonadal development of female rainbow trout (Oncorhynchus mykiss) in a high-altitude cold-water environment. The fish were sampled monthly from January to November and the levels of plasma hormones (estradiol (E2), cortisol and thyroid hormones (THS)) and vitellogenin (VTG) were measured by ELISA. Moreover, the transcriptions of sex-related genes in the ovary, brain, and liver were detected by qRT-PCR. The results showed a seasonal fluctuation of plasma hormones and VTG together with the development of the ovary, which reached a peak from August to October. Similarly, the transcription of hypothalamic gonadotropin-releasing hormone-2 (cgnrh-2), hypothalamic gonadotropin-releasing hormone receptors (gnrhr) and follicle-stimulating hormone (fsh) in the brain varied from January to September, but the highest level was detected in September to November. In addition, the transcription of sex-related genes located in the ovary and liver increased significantly during August to October, accompanied by a continuous increase in the gonadosomatic index (GSI) and a decrease in the hepatosomatic index (HSI). Therefore, plasma hormones and sex-related genes regulate the development and maturation of O. mykiss oocytes with the change in seasons and peaked in November. The results of this study provide a reference for improving the efficiency of the artificial reproduction of O. mykiss.

17β-Trenbolone binds to androgen receptor, decreases number of primordial germ cells, modulates expression of genes related to sexual differentiation, and affects sexual differentiation in zebrafish (Danio rerio)

Exposure to 17β-trenbolone caused a skewed sex ratio in fish. However, the molecular initiating event and key molecular event(s) remain unknown. In this study, zebrafish were exposed to 17β-trenbolone at nominal concentrations of 2 ng/L, 20 ng/L, 200 ng/L, and 2000 ng/L from fertilization to 60 days post fertilization (dpf). First, the sex ratio at 60 dpf was calculated to evaluate adverse outcomes on sexual differentiation. 17β-Trenbolone caused a skewed sex ratio toward males, with intersex individuals observed in the 20 ng/L group and all-male populations found in the 200 ng/L and 2000 ng/L groups. Then, the distribution and number of primordial germ cells, the expression of sex differentiation-related genes, and plasma vitellogenin concentrations were detected in wild-type zebrafish and the EGFP-nanos-3′UTR transgenic line using whole-mount in situ hybridization, real-time PCR, EGFP fluorescence quantification, and enzyme-linked immunosorbent assay. The results indicated that 17β-trenbolone exposure decreased the number of primordial germ cells at 1 dpf and 3 dpf, decreased expression of ovarian differentiation-related genes foxl2 and cyp19a1a at 60 dpf, increased expression of testis differentiation-related genes dmrt1, sox9a, and amh at 60 dpf, and decreased plasma vitellogenin levels at 60 dpf, revealing the key molecular events at different time points involved in affected sexual differentiation by 17β-trenbolone. Finally, molecular docking showed that 17β-trenbolone docked into ligand-binding domain of zebrafish androgen receptor with high binding energy (−3.72 kcal/mol), suggesting that binding to androgen receptor is the molecular initiating event affecting sexual differentiation by 17β-trenbolone. We found that 17β-trenbolone can bind to the zebrafish androgen receptor, decrease the number of primordial germ cells during the early embryonic stage, modulate the expression of genes related to sexual differentiation during gonadal differentiation, and eventually cause a skewed sex ratio toward males in adults.

Insights into the sex-dependent reproductive toxicity of 2-ethylhexyl diphenyl phosphate on zebrafish (Danio rerio)

As a frequently detected organophosphate ester in various environmental media, the toxic effects of 2-ethylhexyl diphenyl phosphate (EHDPHP) on aquatic organisms of different sexes remain unclear. In this study, adult zebrafish were exposed to 2.5, 50, 250 µg/L of EHDPHP for 21 days to investigate its sex-dependent reproductive toxicity and related mechanisms. EHDPHP exposure significantly inhibited the reproduction of zebrafish, evidenced by the reduced spawning of females, depressed growth and development of their offspring. EHDPHP induced greater impacts on the changes of sex hormones and vitellogenin (VTG) in the males than females. For females, the synthesis of testosterone (T) was inhibited because of the down-regulated gnrhr2, gnrhr3, gnrhr4, gnrh3, gnrh2 and er2β in the brain, while 17β-estradiol (E2) increased in 250 µg/L due to up-regulated cyp19a. For males, the promotion of T was directly related to the up-regulation of fshr, 3βhsd, star, cyp11 and cyp17 in the gonad, and eventually led to the increase of E2. The decrease of plasma 11-KT in both sexes could be mainly attributed to the down-regulation of cyp11b and hsd11b. The plasma VTG decreased in females but increased in males, which was in accordance with the down and up regulation of erα and er2β in the females and males, respectively. All these indicated EHDPHP displayed reproductive toxicity on zebrafish in a sex dependent manner. Molecular docking analysis indicated stronger interaction of EHDPHP with the antagonisms of estrogen receptor (ER) and androgen receptor (AR), as well as the agonism of CYP19A1, which further revealed the sex-dependent reproductive toxicity mechanism of EHDPHP. This study highlights the importance of distinguishing males and females in toxicity evaluation of endocrine disruption chemicals.

Targeted and non-targeted analysis of young-of-year smallmouth bass using comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry

Smallmouth bass in the Susquehanna River Basin, Chesapeake Bay Watershed, USA, have been exhibiting clinical signs of disease and reproductive endocrine disruption (e.g., intersex, male plasma vitellogenin) for over fifteen years. Previous histological and targeted chemical analyses have identified infectious agents and pollutants in fish tissues including organic contaminants, mercury, and perfluorinated compounds, but a common causative link for the observed signs of disease across this widespread area has not been determined. This study examines 146 young-of-year smallmouth bass collected from 14 sampling sites in the Susquehanna River Basin, Pennsylvania, USA with varying levels of disease prevalence. Whole fish were extracted by a recently developed modification to the quick, easy, cheap, effective, rugged, and safe extraction method and analyzed by comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry. A targeted analysis was conducted to identify the presence and quantity of 127 known contaminants, including polychlorinated biphenyls, brominated diphenyl ethers, organochlorinated pesticides, and pharmaceutical and personal care products. A non-targeted analysis was conducted on the same data set to identify analytes of interest not included on routine target compound lists. Chromatographic alignment through Statistical Compare (ChromaTOF GC) was followed by Fisher ratio and principal component analysis to reduce the data set from thousands of peaks per sample to a final data set of 65 analytes of interest. Comparisons of these 65 compounds between Normal (no observed health anomalies) and Lesioned (observed health anomaly at time of collection) fish revealed increased levels of three chemical families in Lesioned fish including esters, ketones, and nitrogen containing compounds.