External reproductive structures of male Surfperches (Embiotocidae): Urogenital papilla.

Fishes exhibit a dynamic array of reproductive diversity. Among the various forms of reproduction, internal fertilization has evolved at least 13 times and has been observed in 2-3% of teleost species, half of which are marine. Internal fertilization has led to the evolution of male intromittent organs to aid in sperm transfer during copulation. The intromittent organs of poeciliids have been widely investigated, but little research has focused on the intromittent organs of marine fishes. The surfperches are internally fertilizing, viviparous fishes with a ventrally located papilla. In this article, the external morphology of 19 Embiotocidae species was examined. Additionally, paraffin histology was used to investigate the walleye surfperch (Hyperprosopon argenteum), black perch (Embiotoca jacksoni), and dwarf perch (Micrometrus minimus) papilla to aid in the identification of tissue composition to determine its function. External morphology was documented via macro photography and males were dissected to observe the relationship between the testes, spermatic duct, and papilla. We provide histological evidence that the urogenital papilla is directly connected to the spermatic duct and testis and likely involved in spermatozeugmata transfer. Our research also determined that the papillae were composed of both urinary and reproductive tissues, thus we propose the papilla be referred to as a urogenital papilla. Our study contributes to an overall understanding of intromittent organs of marine fishes, particularly in the surfperches, which has not been previously documented.

Widespread alterations to hypothalamic-pituitary-gonadal (HPG) axis signaling underlie high temperature reproductive inhibition in the eurythermal sheepshead minnow (Cyprinodon variegatus).

Fish experiencing abnormally high or prolonged elevations in temperature can exhibit impaired reproduction, even for species adapted to warm water environments. Such high temperature inhibition of reproduction has been linked to diminished gonadal steroidogenesis, but the mechanisms whereby hypothalamic-pituitary-gonadal (HPG) axis signaling is impacted by high temperature are not fully understood. Here, we characterized differences in HPG status in adult sheepshead minnow (Cyprinodon variegatus), a eurythermal salt marsh and estuarine species of eastern North America, exposed for 14 d to temperatures of 27 °C or 37 °C. Males and females at 37 °C had lower gonadosomatic index (GSI) values compared to fish at 27 °C, and females at 37 °C had fewer spawning capable eggs and lower circulating 17ß-estradiol (E2). Gene transcripts encoding gonadotropin-inhibitory hormone (gnih) and gonadotropin-releasing hormone-3 (gnrh3) were higher in relative abundance in the hypothalamus of both sexes at 37 °C. While pituitary mRNAs for the ß-subunits of follicle-stimulating hormone (fshß) and luteinizing hormone (lhß) were lowered only in males at 37 °C, Fsh and Lh receptor mRNA levels in the gonads were at lower relative levels in both the ovary and testis of fish at 37 °C. Females at 37 °C also showed reduced ovarian mRNA levels for steroid acute regulatory protein (star), P450 side-chain cleavage enzyme (cyp11a1), 3ß-hydroxysteroid dehydrogenase (3ßhsd), 17ß-hydroxysteroid dehydrogenase (hsd17ß3), and ovarian aromatase (cyp19a1a). Females at the higher 37 °C temperature also had a lower liver expression of mRNAs encoding estrogen receptor α (esr1) and several vitellogenin and choriogenin genes, but elevated mRNA levels for hepatic sex hormone-binding globulin (shbg). Our results substantiate prior findings that exposure of fish to high temperature can inhibit gonadal steroidogenesis and oogenesis, and point to declines in reproductive performance emerging from alterations at several levels of HPG axis signaling including increased hypothalamic Gnih expression, depressed gonadal steroidogenesis, and reduced egg yolk and egg envelope protein production in the liver.

Systematic relationships of sympatric pipefishes (Syngnathus spp.): A mismatch between morphological and molecular variation.

Analyses of mitochondrial DNA and morphological variation were performed on specimens of all five currently recognised Syngnathus pipefish species from the eastern Pacific Ocean with type localities currently considered to lie within the Californian marine biogeographic province: kelp pipefish Syngnathus californiensis, bay pipefish S. leptorhynchus, barred pipefish S. auliscus, barcheek pipefish S. exilis and chocolate pipefish S. euchrous. Results consistently differentiate S. auliscus from the other species and fail to distinguish all other specimens as distinct species, as indicated by extensive morphological overlap as well as incomplete lineage sorting and considerably low genetic divergence for 16s and coI genes(<1%). This study presents a taxonomic revision of eastern Pacific Syngnathus spp. and proposes the synonymy of S. leptorhynchus, S. euchrous and S. exilis, under the senior synonym, S. californiensis. There is still a need to study populations of Syngnathus spp. from north and south of the Californian province to assess whether these too are synonyms of the two-species recognised here.

Reproductive Morphology of Oarfish (Regalecus russellii).

Reproduction is a critical aspect of understanding the biology of fishes. Relatively little is known about oarfish (Regalecus russellii) reproduction; however, strandings of dead animals have provided a rare opportunity to investigate the gonadal morphology of four fish: two females and two males. A female collected in June 2015 (4.32 m TL) had bifurcated ovaries 2.14 m in length and 2.14 kg. The gonadosomatic index (GSI) was 11.8% and the fish was determined to be spawning capable/spawning reproductive phase. A female that stranded in Sept. 2015 (5.20 m TL) had bifurcated ovaries 1.43 m in length and 1.28 kg with a GSI of 1.55%. The Sept. female was in a regressing phase of reproduction. A male collected in Aug. 2015 (4.30 m TL) had 64.7-cm-long testes that weighed 40.1 g. The GSI was 0.05% representing a regressing phase of reproduction. A male collected in Nov. 2015 (4.10 m TL) had testes 104.0 cm in length and 467.0 g with a GSI of 0.59%. The Nov. male was in a spawning/spawning capable phase of reproduction. We described ovarian follicles and sperm cells based on size classes and cytological characteristics. We concluded that oarfish are likely batch spawners that undergo periods of regression after a spawning event or season. While this study is not complete with respect to the annual reproductive cycle of oarfish, it markedly contributes to our overall understanding of this rare, mesopelagic fish. Anat Rec, 300:1695-1704, 2017. © 2017 Wiley Periodicals, Inc.

Trenbolone acetate metabolites promote ovarian growth and development in adult Japanese medaka (Oryzias latipes).

Trenbolone acetate, a synthetic androgen, has been used as a growth promoter in beef cattle in the US since 1987. While several teleost studies have investigated the masculinization effects of the metabolite 17ß-trenbolone, few have focused on the reproductive impacts of all three trenbolone acetate (TBA) metabolites including trendione. Adult female medaka (Oryzias latipes) were exposed to TBA metabolites (10, 100, and 1000ng/L) for 14days (n=3). Histological examination revealed that TBA metabolites (1000ng/L) significantly reduced the percentage of primary ovarian follicles and increased the percentage of vitellogenic follicles compared to control fish. 17α-Trenbolone significantly increased whereas trendione reduced whole body levels of estradiol-17ß. Testosterone was significantly reduced by trendione treatment and only the highest dose of 17ß-trenbolone and lowest dose of trendione altered 11-ketotestosterone. Additionally, TBA metabolites may be further broken down and/or metabolized or converted by the animal influencing both sex steroid levels and ovarian development.

Identification and environmental implications of photo-transformation products of trenbolone acetate metabolites.

Despite the widespread use of the anabolic androgen trenbolone acetate (TBA) in animal agriculture, evidence demonstrating the occurrence of TBA metabolites such as 17ß-trenbolone (17ß-TBOH), 17α-trenbolone (17α-TBOH), and trendione (TBO) is relatively scarce, potentially due to rapid transformation processes such as direct photolysis. Therefore, we investigated the phototransformation of TBA metabolites and associated ecological implications by characterizing the photoproducts arising from the direct photolysis of 17ß-TBOH, 17α-TBOH, and TBO and their associated ecotoxicity. LC-HRMS/MS analysis identified a range of hydroxylated products that were no longer photoactive, with primary photoproducts consisting of monohydroxy species and presumptive diastereomers. Also observed were higher-order hydroxylated products probably formed via subsequent reaction of primary photoproducts. NMR analysis confirmed the formation of 12,17-dihydroxy-estra-5(10),9(11),dien-3-one (12-hydroxy-TBOH; 2.2 mg), 10,12,17-trihydroxy-estra-4,9(11),dien-3-one (10,12-dihydroxy-TBOH; 0.7 mg), and a ring-opened 11,12-dialdehyde oxidation product (TBOH-11,12-dialdehyde; 1.0 mg) after irradiation of ∼14 mg of 17ß-trenbolone. Though unconfirmed by NMR, our data suggest that the formation of additional isomeric products may occur, likely due to the reactivity of the unique 4,9,11 conjugated triene structure of trenbolone. In vivo exposure studies employing Japanese medaka (Oryzias latipes) indicate that low concentrations of 17α-TBOH photoproduct mixtures can alter ovarian follicular development, and photoproducts alter whole-body 17ß-estradiol levels. Therefore, direct photolysis yields photoproducts with strong structural similarity to parent steroids, and these photoproducts still retain enough biological activity to elicit observable changes to endocrine function at trace concentrations. These data indicate that environmental transformation processes do not necessarily reduce steroid hormone ecotoxicity.

The effects of the pyrethroid insecticide, bifenthrin, on steroid hormone levels and gonadal development of steelhead (Oncorhynchus mykiss) under hypersaline conditions.

The San Francisco Bay Estuary and Sacramento-San Joaquin Delta (Bay-Delta) is an important breeding and nursery ground for fish. Of particular interest are salmonids that migrate through fresh and saltwater areas polluted with various contaminants including bifenthrin, a widely used pyrethroid insecticide. Male steelhead (Oncorhynchus mykiss) exposed to bifenthrin (0.1 and 1.5µg/L) for two weeks had a lower gonadosomatic index (GSI) in freshwater but were not affected by concurrent bifenthrin exposure and saltwater acclimation. Plasma estradiol-17ß (E2) levels and ovarian follicle diameter of fish exposed to bifenthrin (0.1 and 1.5µg/L) in freshwater significantly increased. Under hypersaline conditions, fish exposed to bifenthrin had significantly reduced E2 levels and smaller follicles, and unhealthy ovarian follicles were observed. Given the occurrence of bifenthrin in surface waters of the Bay Delta, understanding the impact of bifenthrin on wildlife is necessary for improving risk assessments of pyrethroids in this important ecosystem.

Annual and seasonal evaluation of reproductive status in hornyhead turbot at municipal wastewater outfalls in the Southern California Bight.

Treated wastewater effluent containing endocrine-disrupting chemicals is discharged into the coastal waters of the Southern California Bight (SCB) daily. The present study investigated changes in indicators of reproductive health and environmental estrogen exposure in hornyhead turbot (Pleuronichthys verticalis) near wastewater outfalls. Fish were collected from discharge areas, farfield stations, and a reference location in the SCB to examine spatial and temporal patterns. Fish from the Orange County outfall farfield site were younger and less sexually mature than fish from other sites. The sex ratio was skewed in some fish from outfall sites as well as from the Dana Point reference site. However, no consistent pattern in sex ratio was present over time. Low-level induction of vitellogenin was frequently observed in male fish from all sites, suggesting widespread exposure to estrogenic compounds, but did not appear to impact reproductive function as there was no incidence of gonad abnormalities (ova-testis). Analysis of historical hornyhead turbot trawl data indicated that populations are either increasing or stable in the SCB; thus, environmental estrogen exposure was not adversely impacting fish abundance. Additional research is needed to determine the cause of the estrogenic response in hornyhead turbot and whether the source of the estrogenic compounds is a consequence of historical contamination or of ongoing sources or representative of baseline characteristic of this species.

Stage-specific effects of androgens and estradiol-17beta on the development of late primary and early secondary ovarian follicles of coho salmon (Oncorhynchus kisutch) in vitro.

An in vitro system was used to analyze the effects of sex steroids on the development of primary (late perinucleolar stage) and early secondary, previtellogenic (early cortical alveolus stage) ovarian follicles of coho salmon cultured for up to 21 days. Late perinucleolar-stage follicles increased significantly in size after 7 days of treatment with low concentrations of 11-ketotestosterone (11-KT), a nonaromatizable androgen. An androgen receptor antagonist (flutamide) inhibited this growth-promoting effect, and the highest concentration resulted in atresia of follicles, implicating androgens as survival factors at this stage. Testosterone (T) was less effective than 11-KT in promoting growth, but blocking aromatization with exemestane resulted in a growth response similar to that of 11-KT. Estradiol-17beta (E2) had no effect on growth at this stage. After 21 days of culture, E2 was the most potent steroid in increasing the number of follicles containing cortical alveoli and the number of cortical alveoli within those follicles. At the early cortical alveolus stage, low doses of E2 promoted growth and strongly stimulated synthesis of cortical alveoli, actions that were inhibited by an estrogen receptor antagonist (tamoxifen). 11-KT displayed moderate growth-promoting effects, and 11-KT and T stimulated moderate to substantial increases in abundance of cortical alveoli. This study shows that the predominant role of androgens is the promotion of growth of late perinucleolar-stage follicles, while E2 stimulates both the growth and accumulation of cortical alveoli in early cortical alveolus-stage follicles.

Analytical and biological characterization of halogenated gemfibrozil produced through chlorination of wastewater.

The cholesterol-lowering pharmaceutical gemfibrozil is a relevant environmental contaminant because of its frequency of detection in U.S. wastewaters at concentrations which have been shown to disrupt endocrine function in aquatic species. The treatment of gemfibrozil solutions with sodium hypochlorite yielded a 4'-chlorinated gemfibrozil analog (chlorogemfibrozil). In the presence of bromide ion, as is often encountered in municipal wastewater, hypobromous acid generated through a halogen exchange reaction produced an additional 4'-brominated gemfibrozil product (bromogemfibrozil). Standards of chloro- and bromogemfibrozil were synthesized, isolated and characterized using mass spectrometry and NMR spectroscopy. Mass spectrometry was used to follow the in situ halogenation reaction of gemfibrozil in deionized water and wastewater matrices, and to measure levels of gemfibrozil (254 ± 20 ng/L), chlorogemfibrozil (166 ± 121 ng/L), and bromogemfibrozil (50 ± 11 ng/L) in advanced primary wastewater treatment effluent treated by chlorination. Chlorogemfibrozil demonstrated a significant (p < 0.05) reduction in the levels of 11-ketotestosterone at 55.1 µg/L and bromogemfibrozil demonstrated a significant (p < 0.05) reduction in the levels of testosterone at 58.8 µg/L in vivo in Japanese medaka in a 21 day exposure. These results indicated that aqueous exposure to halogenated degradates of gemfibrozil enhanced the antiandrogenicity of the parent compound in a model fish species, demonstrating that chlorination may increase the toxicity of pharmaceutically active compounds in surface water.