Intersex Gonads Research Articles

Phenotypic sorting of individual male and female intersex Cherax quadricarinatus and analysis of molecular differences in the gonadal transcriptome

Cherax quadricarinatus exhibit sexual dimorphism, with males outpacing females in size specification and growth rate. However, there is limited understanding of the molecular mechanisms underlying sex determination and sex differentiation in crustaceans. To study the differences between intersex individuals and normal individuals, this study counted the proportion of intersex individuals in the natural population, collected the proportion of 7 different phenotypes in 200 intersex individuals, and observed the differences in tissue sections. RNA-seq was used to study the different changes in the transcriptome of normal and intersex gonads. The results showed that: the percentage of intersex in the natural population was 1.5 %, and the percentage of different types of intersex ranged from 0.5 % to 22.5 %; the sections revealed that the development of normal ovaries was stagnant at the primary oocyte stage when intersex individuals with ovaries were present; We screened for pathways and genes that may be associated with gonadal development and sex, including ovarian steroid synthesis, estrogen signaling pathway, oocyte meiosis, progesterone-mediated oocyte maturation, etc. Relevant genes including tra2a, dmrta2, ccnb2, foxl2, and smad4. This study provides an important molecular basis for sex determination, sex-controlled breeding, and unisex breeding in red crayfish.

Self-fertilization in a gonochoristic fish yellow catfish

Sex control techniques are in great demand for aquaculture because of the huge benefits of mono-sex production. In a previous study on yellow catfish (Tachysurus fulvidraco), intersex individuals were produced by administrating 17α-methyltestosterone (MT) or aromatase inhibitor letrozole (LZ). The ovarian and testicular portions of intersexual gonads developed well and were completely separated, arousing our interest in producing self-fertilizable fish and clone-like fish populations. In this study, yellow catfish fry were treated with a combination of MT and LZ at a low dose (5 mg kg−1 MT, 300 mg kg−1 LZ) at a low water temperature (27.0 °C, mixed sex-producing temperature, LL group) and a high temperature (33.5 °C, male-producing temperature, HL group), and with a high dose (10 mg kg−1 MT, 600 mg kg−1 LZ) at a low water temperature (LH group), during 12–65 days post-hatching, the critical period of sex differentiation. Both XX and XY intersex were produced with different percentages at the same time through co-treatment with MT and LZ, with or without the help of male-producing temperature. The highest percentage of XY intersex (53% in XY, 36% in XX and XY) and XX intersex (47% in XX, 25% in XX and XY) was produced in LH and HL groups, respectively. Results of gonadal morphology, histology, and sperm dynamic analysis indicate that some XX and XY intersex individuals were capable of self-fertilization through artificial insemination. The reproductive performance of these intersex individuals was then investigated by injecting spawning agents, dissecting gonads, and artificially fertilizing eggs with their own sperm. The XX, XY, and YY genotypic individuals were produced by the self-fertilization of XY intersex individuals. The present study successfully produced high percentages of XX and XY intersex and conducted self-fertilization by mating the eggs and sperm of a single intersex individual. Self-fertilization in fish will show great potential in aquaculture genetics and breeding, for example, generating highly homozygous (or ‘‘clonal’’) genetic material, producing mono-sex populations, and identifying unknown sex determination modes. In particular, the cloning method of producing genetic materials will be greatly beneficial for maintaining advantageous traits through self-fertilization generation after generation.

Otolith shape and elemental signatures provide insights into the connectivity of euryhaline Chelon labrosus inhabiting two close estuaries with different burdens of xenoestrogens in the Southern Bay of Biscay

Intersex gonads have been observed in thicklip grey mullet Chelon labrosus, inhabiting estuaries with high burdens of xenoestrogens in the Southeast Bay of Biscay, but knowledge of population connectivity among estuaries is lacking for this euryhaline fish species. This study investigates the population structure of C. labrosus using otolith shape and elemental signatures of 60 adults (overall length ∼ 38 cm) from two estuaries 21 nautic miles apart, one with a high incidence of intersex condition (Gernika), and the other one pristine (Plentzia). Otolith shape analyses were performed using elliptical Fourier descriptors, while elemental signatures of whole sagittae were obtained by inductively coupled plasma mass spectrophotometry. Univariate and multivariate statistics were applied to determine if otolith signatures show patterns of homogeneity between estuaries. The data indicated significant differences in the otolith shape and elemental composition between mullets of Gernika and Plentzia. Elemental differences were mainly driven by Sr, Li (both higher in Plentzia) and Ba (higher in Gernika). The 98% re-classification success rate obtained from stepwise linear discriminant function analysis suggests that Gernika and Plentzia individuals form separated population units. The limited connectivity between these two close estuaries would indicate a different life history of exposure to chemicals, which might explain the high prevalence of intersex condition in Gernika and its absence in Plenztia.

Steroidogenic potential of the gonad during sex differentiation in the rice field frog Hoplobatrachus rugulosus (Anura: Dicroglossidae).

Prior studies demonstrated that gonadal differentiation in the rice field frog, Hoplobatrachus rugulosus, was of an undifferentiated type since all individuals had ovaries at complete metamorphosis. However, the steroidogenic potential of the gonad is still unknown. In this study, H. rugulosus were obtained by stimulating fertilization in the laboratory under natural light and temperature conditions. The gonads were collected and their steroidogenic potential was evaluated by determining the expression level of messenger RNA (mRNA) encoding for cytochrome P450 17-hydroxylase/C17-20 lyase (CYP17) and cytochrome P450 aromatase (CYP19) using quantitative real-time RT-PCR and the localization of CYP17 mRNA in tissues by in situ hybridization. The CYP17 mRNA levels in males at 4-11 weeks postmetamorphosis were higher than in female and intersex gonads. This corresponded to their localization in the gonadal tissues, where CYP17 signals were specifically detected in the Leydig cells of the testis at 5-16 weeks postmetamorphosis but was undetectable in all ovary samples. The CYP19 mRNA levels in females at 4-11 weeks postmetamorphosis was higher than in male and intersex gonads, which corresponded with gonadal development, indicating the potential steroidogenic function of the ovary. Based on the present results, the role of CYP17 and CYP19 mRNA in sex differentiation in H. rugulosus may occur after gonadal sex differentiation and the steroidogenic potential of the gonads exhibited a sexual dimorphic pattern. These results provide a crucial basis for further research on the developmental biology in anuran species.

Reproductive endocrine disruption and gonadal intersex induction in male Japanese medaka chronically exposed to betamethasone at environmentally relevant levels

The broad utilization of betamethasone in medical treatments may pose a significant ecotoxicological risk to aquatic organisms, yet its potential reproductive toxicity remains unclear. The present study examined the impacts of environmental exposure on male reproduction using Japanese medaka (Oryzias latipes). After 110 days of betamethasone exposure at environmentally relevant concentrations (0, 20 and 200 ng/L), LH/FSH synthesis and release in the pituitary was inhibited, and the production of sex hormones and their signaling pathways in the gonads of male medaka were greatly influenced. This synthetic glucocorticoid restrained testosterone (T) synthesis and gave rise to a significant increase in E2/T and E2/11-KT ratios. Furthermore, chronic betamethasone exposure (20 and 200 ng/L) led to the suppression of androgen receptor (AR) signaling and enhancement of estrogen receptors (ERs) signaling. An increase in hepatic vitellogenin contents was also detected, and testicular oocytes were observed in both 20 and 200 ng/L betamethasone-treated groups. It showed that 20 and 200 ng/L betamethasone could induce male feminization and even intersex, triggering abnormal spermatogenesis in medaka males. With its adverse effects on male fertility, betamethasone could potentially influence the fishery productivity and population dynamics in aquatic ecosystems.

Integrative study of the reproductive biology and growth of Acestrorhynchus pantaneiro Menezes, 1992 (Characiformes, Acestrorhynchidae).

We studied the reproductive strategy, sexual system and growth of dientudo paraguayo Acestrorhynchus pantaneiro. After 2 years of monitoring in shallow areas of a floodplain lake from the lower Paraná basin (Argentina), it was evidenced that water temperature modulated gonadal maturation, but it wasthe river water level the synchronising stimulus that triggered spawning. This species exhibited a single annual breeding period from October to January, with most spawning activity in November. According to the von Bertalanffy growth curve, fish would reach autumn to winter months with LS of ~120 mm, already mature males. The first mature females were found at LS of 210 mm, becoming sexually mature between the second and third breeding seasons. This is the first integrative study that includes the body-length frequency distribution and sex differential size at first maturity and growth, and reports the presence of intersex gonads questioning its sexualpattern from gonochoristic to sequential hermaphrodite species. The sexual pattern, the multiple spawning behaviour and a medium to high absolute fecundity support the opportunistic andinvasive behaviorobserved in previous contributions for this characiform species.

Effects of Anthropic Pollutants Identified in Pampas Lakes on the Development and Reproduction of Pejerrey Fish Odontesthes bonariensis

Anthropic activities can seriously affect the health of the organisms inhabiting them, and the observation of any alteration in the reproduction of fish could be associated with the presence of endocrine disruptors. In this manuscript we have collected information on the adverse effects of pollutants (heavy metals, environmental steroids, and agrochemicals), present in Chascomús lake, Argentina, either at environmentally relevant and pharmacological concentrations on reproduction, embryonic development, and larval survival of pejerrey fish Odontesthes bonariensis. During development, it has been reported that 17β-estradiol (E2) feminized and reduced larval survival, while 17α-ethinyl-estradiol (EE2) not only feminized but also affected both embryo and larval survival. In adult male fish, treatments with EE2 and E2 + EE2 were able to increase mRNA abundance of gnrh3 and cyp19a1b and decreased those of gonadotropin receptors (fshr and lhcgr). Heavy metals such as cadmium, chromium, and copper negatively affected sperm quality, diminishing the motility. Also, a decrease in the percentage of hatching rate and larval survival was also observed with the same metals, highlighting zinc as the most detrimental metal. Furthermore, all these metals altered the expression of hypothalamic and pituitary genes related to reproduction in male pejerrey (gnrh1,2,3; cyp19a1b; fshb; lhb; fshr and, lhcgr). Moreover, in all cases pyknotic cells, corresponding to the degeneration of the germ cells, were observed in the testes of exposed fish. For agrochemicals, exposure of male pejerrey to environmental concentrations of glyphosate did not cause alterations on the endocrine reproductive axis. However, male pejerrey with gonadal abnormalities such as the presence of intersex (testis-ova) gonads were found in other Pampa´s lakes with high concentrations of atrazine and glyphosate associated with soybean and corn crops near their coasts. These types of studies demonstrate that pejerrey, an endemic species with economic importance inhabiting the Pampas shallow lakes, can be used as a sentinel species. It should be noted that increased pollution of aquatic ecosystems and the effects on the reproduction of organisms can lead to a decline in fish populations worldwide. Which, added to overfishing and other external factors such as global warming, could cause an eventual extinction of an emblematic species.

Identification, characterization and functional analysis of gonadal long noncoding RNAs in a protogynous hermaphroditic teleost fish, the ricefield eel (Monopterus albus)

BackgroundAn increasing number of long noncoding RNAs (lncRNAs) have been found to play important roles in sex differentiation and gonad development by regulating gene expression at the epigenetic, transcriptional and posttranscriptional levels. The ricefield eel, Monopterus albus, is a protogynous hermaphroditic fish that undergoes a sequential sex change from female to male. However, the roles of lncRNA in the sex change is unclear.ResultsHerein, we performed RNA sequencing to analyse lncRNA expression patterns in five different stages of M. albus development to investigate the roles of lncRNAs in the sex change process. A total of 12,746 lncRNAs (1503 known lncRNAs and 11,243 new lncRNAs) and 2901 differentially expressed lncRNAs (DE-lncRNAs) were identified in the gonads. The target genes of the DE-lncRNAs included foxo1, foxm1, smad3, foxr1, camk4, ar and tgfb3, which were mainly enriched in signalling pathways related to gonadal development, such as the insulin signalling pathway, MAPK signalling pathway, and calcium signalling pathway. We selected 5 highly expressed DE-lncRNAs (LOC109952131, LOC109953466, LOC109954337, LOC109954360 and LOC109958454) for full length amplification and expression pattern verification. They were all expressed at higher levels in ovaries and intersex gonads than in testes, and exhibited specific time-dependent expression in ovarian tissue incubated with follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG). The results of quantitative real-time PCR (qRT-PCR) analysis and a dual-luciferase assay showed that znf207, as the gene targeted by LOC109958454, was expressed in multiple tissues and gonadal developmental stages of M. albus, and its expression was also inhibited by the hormones FSH and hCG.ConclusionsThese results provide new insights into the role of lncRNAs in gonad development, especially regarding natural sex changes in fish, which will be useful for enhancing our understanding of sequential hermaphroditism and sex changes in the ricefield eel (M. albus) and other teleosts.

Sexual development dysgenesis in interspecific hybrids of Medaka fish

Fish are amongst vertebrates the group with the highest diversity of known sex-determining genes. Particularly, the genus Oryzias is a suitable taxon to understand how different sex determination genetic networks evolved in closely related species. Two closely related species, O. latipes and O. curvinotus, do not only share the same XX/XY sex chromosome system, but also the same male sex-determining gene, dmrt1bY. We performed whole mRNA transcriptomes and morphology analyses of the gonads of hybrids resulting from reciprocal crosses between O. latipes and O. curvinotus. XY male hybrids, presenting meiotic arrest and no production of sperm were sterile, and about 30% of the XY hybrids underwent male-to-female sex reversal. Both XX and XY hybrid females exhibited reduced fertility and developed ovotestis while aging. Transcriptome data showed that male-related genes are upregulated in the XX and XY female hybrids. The transcriptomes of both types of female and of the male gonads are characterized by upregulation of meiosis and germ cell differentiation genes. Differences in the parental species in the downstream pathways of sexual development could explain sex reversal, sterility, and the development of intersex gonads in the hybrids. We hypothesize that male-to-female sex reversal may be connected to a different development time between species at which dmrt1bY expression starts. Our results provide molecular clues for the proximate mechanisms of hybrid incompatibility and Haldane’s rule.

ELUCIDATING THE GONADAL DEVELOPMENT AND SEX DIFFERENTIATION OF THE RICE PADDY EEL, Monopterus albus (Zuiew, 1993), BASED ON GONADAL HISTOMORPHOLOGY AND STEROID HORMONE LEVELS

The establishment and survival of the population of species in their breeding ground can be attributed to the species’ reproductive characteristic and capacity. The Monopterus albus species is an introduced fish in the Philippines. It is a protogynous hermaphrodite which has a natural ability for sex reversal. This study describes the histomorphology of the different stages of gonadal maturation and determines the concentration levels of the steroid hormones 17ß-estradiol (E2) and testosterone (T) in the blood plasma. These aim to a further understanding on the gonadal development and sex differentiation of the rice paddy eel as a hermaphroditic fish. Fish samples were collected randomly from farm ponds located in three provinces within the island of Luzon, Philippines. The sexual identity of the fish was categorized based on the presence or absence of ovarian and testicular tissues. Histological analysis of the gonad showed that immature, maturing-matured female gonad exhibited ovarian tissues of varying stages. The intersex gonad showed the existence of both ovarian and testicular tissues. Enzyme-linked immunosorbent assay (ELISA) was used to determine the concentration levels of gonadal steroid hormones. Differences in the concentration levels of the two hormones were evident across all the stages of gonad maturation. The functional role of these hormones in vitellogenesis and spermatogenesis leads to the development of this sex-changing gonads. This reproductive characteristic contributes to our understanding of the reproductive success of hermaphroditic fish.

Bulk and single-cell RNA-seq reveal the sexually dimorphic expression pattern of dmrtb1 in zig-zag eel (Mastacembelus armatus)

Sequential hermaphroditism, whereby the sex of one individual changes during its development, has been described in many fish species. Yet, the genetic regulation of this dynamic process of sexual differentiation is not well understood. Here, we report the characterization of intersexual differences in an aquacultural fish, the zig-zag eel (Mastacembelus armatus), based on 4 years of artificial breeding. All M. armatus individuals initially developed as females, with approximately half of them then becoming males after going through an intersex phase. A DM-domain candidate gene, dmrtb1, was identified from gonadal transcriptome data screening. The expression of dmrtb1 was restricted to testis, similar to dmrt1, which is one of the conserved sex-determining genes in vertebrates. Interestingly, dmrtb1 was expressed at the onset of sex change (early stage), and gradually increased in expression in the intersex gonads until the late stage of differentiation. Dmrtb1 was also expressed in the seminiferous lobules containing cysts with germ cells. A mixed cell population from ovotestis was identified by single-cell RNA sequencing, and included mitotic fetal germ cells, meiotic prophase fetal germ cells, and gonad endothelial cells. Spermatocyte and oocyte marker genes were both enriched in the same gonadal cell population, indicating this cell population might have the potential of bisexual differentiation. The correlation between dmrtb1 expression and sex-change development suggests M. armatus as an emerging model to study the molecular mechanism of sexual differentiation in fish. Such information will be vital for promoting the conservation of this species in the wild as well as for developing a sustainable aquacultural industry.

Production of YY males through self-fertilization of an occasional hermaphrodite in Lanzhou catfish (Silurus lanzhouensis)

Lanzhou catfish (Silurus lanzhouensis), an important commercial fish in China, takes three years to be sexually mature and males display faster growth performance than females. Thus, shortening of breeding cycle and production of all-male population in Lanzhou catfish have great significance in aquaculture. Here, we first developed an optimal method for artificial gynogenesis, through which the male heterogametic XX/XY sex determination system was revealed in Lanzhou catfish. Subsequently, sex-specific markers were identified via 2b-RAD-seq and verified in artificial breeding and wild populations. Intriguingly, a hermaphrodite Lanzhou catfish with XY genotype was occasionally obtained, whose gonad contained both mature ovarian and testicular tissues. The eggs and sperm from the intersexual gonad were used for artificial fertilization to establish a self-fertilization family, which contained 21 XX females, 25 XY males, and 12 YY males in the offspring. And the YY males are able to mate with XX females to produce all-male progenies with XY genotype. Compared with the conventional way of expending two-generation time, self-fertilization of the XY hermaphrodite saved one-generation time to produce YY males. Therefore, this study not only reveals XX/XY sex determination system in Lanzhou catfish, but also directly obtains YY males for future production of all-male population in aquaculture.

Cloning and characterization of rec8 gene in orange-spotted grouper (Epinephelus coioides) and Dmrt1 regulation of rec8 promoter activity

Meiosis is a specialized type of cell division critical for gamete production during sexual reproduction in eukaryotes. The meiotic recombination protein Rec8 has been identified as an important factor in germ cell meiotic initiation in vertebrates; however, its equivalent role in teleosts is poorly characterized. In this study, we cloned and sequenced the rec8 gene from orange-spotted grouper (Epinephelus coioides). The cDNA sequence consisted of 2244 base pairs (bp), including a 5' untranslated region (UTR) of 198bp and a 3'UTR of 284bp. The open reading frame of grouper rec8 was 1752bp, encoding 584 amino acids. Expression levels of rec8 were higher in the ovary, intersex gonad, and testis. A neighbor-joining phylogenetic tree based on the deduced amino acid sequence indicated a common origin for grouper and other teleost rec8 molecules. Immunohistochemistry using a polyclonal anti-Rec8 antibody localized the protein in the oogonia and primary oocytes in the ovary and in spermatogonia and spermatocytes in the intersex gonad and testis, suggesting that Rec8 may play an important role in the meiotic division and the development of grouper germ cells. In addition, we found that the transcription factor Dmrt1 increased rec8 promoter activity through the second binding site, based on dual-luciferase assays. Together, these results suggest that Rec8 plays a crucial role in meiosis and may be regulated by Dmrt1 to affect meiosis in groupers.

A Transient Hermaphroditic Stage in Early Male Gonadal Development in Little Yellow Croaker, Larimichthys polyactis.

Animal taxa show remarkable variability in sexual reproduction, where separate sexes, or gonochorism, is thought to have evolved from hermaphroditism for most cases. Hermaphroditism accounts for 5% in animals, and sequential hermaphroditism has been found in teleost. In this study, we characterized a novel form of the transient hermaphroditic stage in little yellow croaker (Larimichthys polyactis) during early gonadal development. The ovary and testis were indistinguishable from 7 to 40 days post-hatching (dph). Morphological and histological examinations revealed an intersex stage of male gonads between 43 and 80 dph, which consist of germ cells, somatic cells, efferent duct, and early primary oocytes (EPOs). These EPOs in testis degenerate completely by 90 dph through apoptosis yet can be rescued by exogenous 17-β-estradiol. Male germ cells enter the mitotic flourishing stage before meiosis is initiated at 180 dph, and they undergo normal spermatogenesis to produce functional sperms. This transient hermaphroditic stage is male-specific, and the ovary development appears to be normal in females. This developmental pattern is not found in the sister species Larimichthys crocea or any other closely related species. Further examinations of serum hormone levels indicate that the absence of 11-ketotestosterone and elevated levels of 17-β-estradiol delineate the male intersex gonad stage, providing mechanistic insights on this unique phenomenon. Our research is the first report on male-specific transient hermaphroditism and will advance the current understanding of fish reproductive biology. This unique gonadal development pattern can serve as a useful model for studying the evolutionary relationship between hermaphroditism and gonochorism, as well as teleost sex determination and differentiation strategies.

Pattern of Gonadal Sex Differentiation in the Rice Field Frog Hoplobatrachus rugulosus (Anura: Dicroglossidae).

Sex differentiation during gonadal development is diversified among anuran amphibian species. In this study, the anuran experimental species Hoplobatrachus rugulosus was examined. The pattern of gonadal sex differentiation was observed by morphological and histological approaches. The gonad was observed morphologically at Gosner stage 33, while distinct testis and ovary were evident from 3-4 weeks after metamorphosis ended. Histological analysis showed that genital ridge formation began at stage 25 and ovarian differentiation began at stage 36. The developing ovary appeared with numerous primary oogonia, which developed into oocytes, while the medulla regressed to form an ovarian cavity. During metamorphosis, only an ovary was observed. Testicular differentiation seemed to begin later, during the first week after metamorphosis, and occurred via an intersex condition. The intersex gonads contained developing testicular tissue with both normal and atretic oocytes. The fully developed testis was first identified at 6 weeks after metamorphosis. Comparing the times of gonadal differentiation and somatic development revealed that the ovary exhibited a basic rate of differentiation while the testis exhibited a retarded one. These results establish that males of this species develop later than do females, and the testis develops through an intersex gonad, as is evident from its seminiferous cord formation, the presence of testis-ova, and atretic oocytes in the tissue. Thus, the pattern of gonadal sex differentiation in H. rugulosus is an undifferentiated type, in which only female gonads are observed during metamorphosis and intersex and male gonads are observed later. These results are crucial for further research on the sexual development of anurans.

Gonadal development and intersex condition of marbled lungfish, Protopterus aethiopicus (Heckel, 1851), in contaminated sites in Lake Victoria, Uganda

The first evidence of the impact of environmental contamination on populations of Protopterus aethiopicus (Heckel, 1851) in terms of body indices, cellular development (histology) and intersex condition in Lake Victoria, Uganda is provided. Specimens were obtained from the more polluted swampy areas receiving wastes from residential and industrial settings (Ggaba, n = 11; Port Bell, n = 10; and Jinja, n = 7), and rural, less polluted areas (Bukakata, n = 3; Kasensero, n = 4; and Bale, n = 2), and gonadosomatic indices, growth condition factor and the morphometric body characteristics were determined. Fish specimens in less polluted sites had better condition and shapes, regardless of sex, compared with those in more polluted sites of the lake. Most lungfish collected had a total length of 45–55 cm. Irrespective of sex of fish and level of pollution at collection sites, samples (n = 26) collected inshore (1 063.42 g ± 109.32 SE) were heavier than those collected (n = 11) offshore (680.09 g ± 108.85 SE), F (1, 35) = 6.288, p = 0.019. Mean values of gonadosomatic indices were significantly higher in less polluted sites, compared with chemically contaminated urban sites (F (5, 31) = 2.783, p = 0.034), suggesting better growth performance in the former. Spermatogenic cell development progressed as spermatogonia, spermatocytes, spermatids and spermatozoa among males. For oogenic cells, development was through chromatin nucleolar, perinucleolar, cortical alveolar, early vitellogenic and late vitellogenic oocytes. Histological examinations revealed group asynchronous gonadal development and intersex condition among P. aethiopicus populations in Lake Victoria.

Potential Link between Equol Pollution and Field-Observed Intersex in Wild So-iuy Mullets (Mugil soiuy)

Gonadal intersex has been observed in wild fishes and is attributed to endocrine-disrupting chemicals but the specific causes remain controversial. Here, a forensic analysis utilizing field and laboratory studies was conducted to explore the causal agent(s). In a 2008-2009 survey of Liaodong Bay, China, 20.7-33.3% incidences of gonadal intersex were observed in male so-iuy mullets (Mugil soiuy), a wild sentinel fish species. Steroidal estrogen (estrone, 17β-estradiol, estriol, and ethinylestradiol) and phytoestrogen (equol) were detected in seawater where the fishes were collected with median concentrations of 0.42 ng/L (0.02-1.42 ng/L) E2 equivalent (EEQ-E2) and 22.81 ng/L (0.10-155.99 ng/L) equol. A probabilistic model was used to evaluate the ecological risk of these estrogenic chemicals based on their distribution in the field and dose-response relationship from the laboratory surrogate Japanese medaka (Oryzias latipes) fish. The probability of the incidences of gonadal intersex due to equol exposure was estimated to be 13.5 ± 12.1%, which is considerably higher than that for EEQ-E2, (7.2 ± 68.8) × 10-4. The agonistic activity of equol to the estrogen receptor α of so-iuy mullets was 3.5-fold higher than that to the estrogen receptor α of Japanese medaka, indicating that equol shows a stronger potential for inducing intersex in so-iuy mullets than in medaka. These results demonstrate that equol, rather than steroid estrogens, is a more likely causal agent for the field-observed intersex in male wild so-iuy mullets.

Gonad metabolomics and blood biochemical analysis reveal differences associated with testicular oocytes in wild largemouth bass (Micropterus salmoides)

Adverse reproductive effects associated with gonadal intersex among freshwater fish could hold considerable implications for population sustainability. Presence of testicular oocytes (TO) is the most common form of intersex and is widespread among centrarchids (sunfishes) of North America and other freshwater teleosts. Placing TO within the toxicological context of adverse outcome pathways (AOPs) to assess ecological risk is a priority for ecotoxicologists due to the association of TO with harmful chemical exposure and adverse reproductive effects in some cases. However, key event relationships between EDC exposure, incidence of TO, and apical outcomes have yet to be fully elucidated - in part due to a lack of knowledge of relationships between intersex gonad physiology and fish health. Understanding the physiological status of intersex fish is critical to assess ecological risk, understand mechanisms of induction, and to establish biomarkers of intersex in fish. In the present study, features of gonad metabolite profiles associated with TO in largemouth bass (LMB, Micropterus salmoides) from an impoundment in Georgia (USA) were determined using GC-MS-based metabolomics. Clinical blood biochemical screens were used to evaluate markers of fish health associated with TO. Results suggest that physiological changes in energy expenditure as well as relatively 'feminized' gonad lipid and protein metabolism may be related to the occurrence of TO in male LMB, and highlight the need to understand relationships between intersex and physical stressors such as elevated temperature and hypoxia. These results provide novel insight to AOPs associated with TO and identify candidate analytes for biomarker discovery.

Natural sex change in mature protogynous orange-spotted grouper (Epinephelus coioides): gonadal restructuring, sex hormone shifts and gene profiles.

Sexual patterns of teleosts are extremely diverse and include both gonochorism and hermaphroditism. As a protogynous hermaphroditic fish, all orange-spotted groupers (Epinephelus coioides) develop directly into females, and some individuals change sex to become functional males later in life. This study investigated gonadal restructuring, shifts in sex hormone levels and gene profiles of cultured mature female groupers during the first (main) breeding season of 2019 in Huizhou, China (22° 42' 02.6″ N, 114° 32' 10.1″ E). Analysis of gonadal restructuring revealed that females with pre-vitellogenic ovaries underwent vitellogenesis, spawning and regression and then returned to the pre-vitellogenic stage in the late breeding season, at which point some changed sex to become males via the intersex gonad stage. A significant decrease in the level of serum 17β-estradiol (E2) was observed during ovary regression but not during sex change, whereas serum 11-ketotestosterone (11-KT) concentrations increased significantly during sex change with the highest concentration in newly developed males. Consistent with serum hormone changes, a significant decrease in cyp19a1a expression was observed during ovary regression but not during sex change, whereas the expression of cyp11c1 and hsd11b2 increased significantly during sex change. Interestingly, hsd11b2 but not cyp11c1 was significantly upregulated from the pre-vitellogenic ovary stage to the early intersex gonad stage. These results suggest that a decrease in serum E2 concentration and downregulation of cyp19a1a expression are not necessary to trigger the female-to-male transformation, whereas increased 11-KT concentration and upregulation of hsd11b2 expression may be key events for the initiation of sex change in the orange-spotted grouper.

SAT-732 Historical Occurrence of Intersex in Largemouth Bass (Micropterus Salmoides) and Smallmouth Bass (Micropterus Dolomieu)

The widespread occurrence of gonadal intersex (presence of testicular oocytes) has been recently reported in populations of Largemouth Bass (Micropterus salmoides) and Smallmouth Bass Micropterus dolomieu) from populations across North America. To evaluate the historical occurrence of intersex in bass species, gonads were examined visually and histologically from Largemouth Bass and Smallmouth Bass from museum specimens collected from 16 States. These fishes were collected between 1823-1965, before the widespread discharge of estrogenic chemicals to surface waters via wastewater effluents. All females examined had normal ovaries. Intersex gonads were identified in 17.8 % of male Largemouth Bass, and in 14.2% of male Smallmouth Bass. The intensity of testicular oocytes in some males was as great as observed since 2000. These results indicate that the occurrence of this form of intersex in these bass species is not a recent phenomenon.