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.

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.

Effects of waterborne exposure to 17ß-estradiol and 4-tert-octylphenol on early life stages of the South American cichlid fish Cichlasoma dimerus.

Estrogenic chemicals are often detected in the aquatic environment and can negatively affect animal development and reproduction. In teleost fishes, the hormonal regulation during a critical period of larval development has a strong influence on gonadal sex differentiation; thus this process may be affected by the exposure to environmental estrogens. In this study, we first assessed the lethal acute toxicity of the natural estrogen 17ß-estradiol (E2) and the weaker estrogen mimics 4-tert-octylphenol (OP) and 4-nonylphenol (NP) on larval stages of the South American cichlid fish Cichlasoma dimerus. In a further experiment, we analyzed the effects of chronic waterborne exposure to E2 and OP on gonad development and sex differentiation. Exposure to high concentrations of E2 had a pronounced feminizing effect directing sex differentiation towards ovarian development, while testis development was inhibited at a lower, environmentally relevant concentration. Among OP-exposed fish, 15-38.5% of the males exhibited testicular oocytes (TOs), a commonly reported biomarker of estrogenic exposure. However, since TOs were also recorded in control males and the proportion of males with TOs was not significantly higher in OP treatments, their occurrence could not be attributed to OP exposure. In addition, TOs did not seem to impair male gonad development and functionality since normal spermatogenesis was observed in testes of OP-treated fish. These results indicate that E2 occurring in the South American aquatic environment may affect male reproductive development and pose a risk for wild C. dimerus, especially under prolonged exposure, while the effects of weaker xenoestrogens such as OP would be negligible for gonad development in this species. As illustrated by this study, the natural occurrence of TOs indicates that conclusions concerning the causes of this phenomenon must be drawn with care.

Testicular oocytes in smallmouth bass in northeastern Minnesota in relation to varying levels of human activity.

Testicular oocytes (TOs) have been found in black bass (Micropterus spp.) from many locations in North America. The presence of TOs is often assumed to imply exposure to estrogenic endocrine disrupting compounds (EDCs); however, a definitive causal relationship has yet to be established, and TO prevalence is not consistently low in fish from areas lacking evident EDC sources. This might indicate any of a number of situations: 1) unknown or unidentified EDCs or EDC sources, 2) induction of TOs by other stressors, or 3) testicular oocytes occurring spontaneously during normal development. In the present study, we analyzed TO occurrence in smallmouth bass (Micropterus dolomieu) from 8 populations in northeastern Minnesota watersheds with differing degrees of human development and, hence, presumed likelihood of exposure to anthropogenic chemicals. Three watersheds were categorized as moderately developed, based on the presence of municipal wastewater discharges and higher human population density (4-81 per km2 ), and 5 watersheds were minimally developed, with very low human population density (0-1 per km2 ) and minimal built environment. Testicular tissues from mature fish were evaluated using a semiquantitative method that estimated TO density, normalized by cross-sectional area. Testicular oocyte prevalence and density among populations from moderately developed watersheds was higher than in populations from minimally developed watersheds. However, TO prevalence was unexpectedly high and variable (7-43%) in some populations from minimally developed watersheds, and only weak evidence was found for a relationship between TO density and watershed development, suggesting alternative or more complex explanations for TO presence in smallmouth bass from this region. Environ Toxicol Chem 2017;36:3424-3435. © 2017 SETAC.

Gonadal intersex in smallmouth bass Micropterus dolomieu from northern Indiana with correlations to molecular biomarkers and anthropogenic chemicals.

Over the past decade, studies have shown that exposure to endocrine disrupting chemicals (EDCs) can cause gonadal intersex in fish. Smallmouth bass (Micropterus dolomieu) males appear to be highly susceptible to developing testicular oocytes (TO), the most prevalent form of gonadal intersex, as observed in various areas across the U.S. In this study, prevalence and severity of TO was quantified for smallmouth bass sampled from the St. Joseph River in northern Indiana, intersex biomarkers were developed, and association between TO prevalence and organic contaminants were explored. At some sites, TO prevalence reached maximum levels before decreasing significantly after the spawning season. We examined the relationship between TO presence and expression of gonadal and liver genes involved in sex differentiation and reproductive functions (esr1, esr2, foxl2, fshr, star, lhr and vtg). We found that vitellogenin (vtg) transcript levels were significantly higher in the liver of males with TO, but only when sampled during the spawning season. Further, we identified a positive correlation between plasma VTG levels and vtg transcript levels, suggesting its use as a non-destructive biomarker of TO in this species. Finally, we evaluated 43 contaminants in surface water at representative sites using passive sampling to look for contaminants with possible links to the observed TO prevalence. No quantifiable levels of estrogens or other commonly agreed upon EDCs such as the bisphenols were observed in our contaminant assessment; however, we did find high levels of herbicides as well as consistent quantifiable levels of PFOS, PFOA, and triclosan in the watershed where high TO prevalence was exhibited. Our findings suggest that the observed TO prevalence may be the result of exposures to mixtures of nonsteroidal EDCs.

Testicular oocytes in MRL/MpJ mice possess similar morphological, genetic, and functional characteristics to ovarian oocytes.

In general, mammalian males produce only spermatozoa in their testes and females produce only oocytes in their ovaries. However, newborn MRL/MpJ male mice produce oocytes within their testes. In this study, we examined the initiation and progression of oogenesis in fetal and neonatal MRL/MpJ mouse testes and evaluated the characteristics of testicular oocytes. Germ cells with positive reactions to oogenesis markers such as NOBOX oogenesis homeobox and synaptonemal complex protein 3 were observed in the MRL/MpJ fetal testes on embryonic day 18.5. These fetal testicular oocytes possessed maternal-specific methylation patterns of histone and DNA. The level of DNA methylation was still low in postnatal testicular oocytes at day 14 after birth. Additionally, the postnatal testicular oocytes contained both X and Y chromosomes and had the ability to fuse with sperm. These results suggest that some XY germ cells in fetal testes of MRL/MpJ mice enter meiosis prematurely, undergo oogenesis, and differentiate into oocytes. In addition, MRL/MpJ testicular oocytes have the ability to carry on oogenesis before and shortly after birth until they obtain some of the morphological, epigenetic, and functional characteristics of oocytes.

Intersex (testicular oocytes) in largemouth bass (Micropterus salmoides) on the Delmarva Peninsula, USA.

The authors describe the prevalence and severity of intersex in the form of testicular oocytes in largemouth bass (Micropterus salmoides) collected over a 5-yr period from a variety of surface waters on the Delmarva Peninsula, USA, a region dominated by poultry production and agricultural land use. During a survey from 2005 to 2007 of approximately 200 male specimens representing 6 fish and 2 frog species collected from numerous small-order streams on Delmarva, intersex was observed in only largemouth bass (system-wide prevalence 17%). During 2008 and 2009, testicular oocytes were encountered in male largemouth bass from 6 lakes and 1 large river system, with prevalence ranging from 33% to 88% (weighted arithmetic mean, 57%). The prevalence of testicular oocytes in largemouth bass from Delmarva lakes was comparable to the highest levels reported in a national US Geological Survey reconnaissance of this species, which also occurred in regions of the Atlantic coastal plain with intensive row-crop and animal agriculture. To the authors' knowledge, the present study represents the first report in the peer-reviewed scientific literature of testicular oocytes in fish on the Delmarva Peninsula.