Period Of Sexual Differentiation Research Articles

Completely blocking the feminization by replacing Limnodilus spp. with Artemia salina to feeding XY Zig-zag eel (Mastacembelus armatus)

Zig-zag eel (Mastacembelus armatus), with male growing faster and larger than female, is a popular farmed fish in China due to its delicious taste and rich nutritional value. The culture of mono-sex M. armatus has great value and is the best choice for aquaculture industry. However, the high feminization rate in aquaculture severely limits the application of sex control in the breeding process of M. armatus. In this study, by analyzing the early gonadal development and sex differentiation process, as well as the expression profiles of male and female pathway genes in the early XX and XY gonads, we determined that the critical period for sex differentiation and gonadal differentiation in M. armatus was 10–30 dpf. In addition, we confirmed for the first time that the feminization of M. armatus is related to feeding Limnodilus spp., as only Limnodilus spp. are fed during 10–30 dpf. Replacing Limnodilus spp. with Artemia salina to feed M. armatus fry can completely block the feminization. These results lay a solid foundation for understanding the sex determination mechanism of M. armatus, and the method of blocking the feminization of M. armatus can be used for the development of all-male breeding.

Characteristics of testis development in autotetraploid fish

Fertile autotetraploid fish are an excellent germplasm resource for producing improved triploids. However, males produce more diploid gametes than females during production, which means that males are more valuable in application. To better understand the testis development of autotetraploid fish (4nRR) derived from the distant hybridization of Carassius auratus red var. (RCC, ♀) × Megalobrama amblycephala (BSB, ♂), all-male autotetraploid fish (AM4nRR) were generated. In this study, the tissue structure and transcriptome changes in different periods of gonadal development of AM4nRR were analyzed. Histological sectioning revealed a small number of gonium at 45 days post-hatching (dph), the number of germ cells greatly increased during 65–95 dph, and primary spermatocytes and secondary spermatocytes were observed at 105 dph. Meanwhile, white semen could be extruded from most individuals at 120 dph, and the DNA content of sperm from AM4nRR was approximately twice that from RCC as determined by flow cytometry. These results indicated that AM4nRR reached sexual maturity at 120 dph and produced diploid sperm. In addition, a total of 149.55 Gb of clean data and 56,188 unigenes were obtained from the testicular tissue of AM4nRR at different developmental periods (25 dph, 35 dph, 40 dph, 45 dph, 65 dph, 95 dph and 120 dph). Differential expression analysis showed that the number of differentially expressed genes (DEGs) among the three periods of 40 dph, 45 dph, and 65 dph was the lowest (72–275). However, the expression levels of partial DEGs at 45 dph changed abruptly compared with those at 40 dph and 65 dph according to hierarchical clustering analysis, which was consistent with the expression characteristics of gonadal development-related genes at the early period of sex differentiation. These results suggested that 40–65 dph might be a critical period for the initiation of testis development in autotetraploid fish. These findings not only provide a new gamete resource for the large-scale preparation of improved triploids but also provide a basis for further study of sex differentiation in autotetraploid fish.

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.

The fusion gene hsf5-rnf43 in Nile tilapia: A potential regulator in the maintenance of testis function and sexual differentiation

We identified that the genes heat shock transcription factor 5 (hsf5) and ring finger protein 43 (rnf43) happened fusion in Nile tilapia (Oreochromis niloticus), called hsf5-rnf43, and provided the characteristic and functional analysis of hsf5-rnf43 gene in fish for the first time. Analysis of spatiotemporal expression showed that hsf5-rnf43 was specifically expressed in the testis and located in primary spermatocytes of adult Nile tilapia and gradually increased during testis development from 5 to 180days after hatching. We also found DNA methylation regulated sex-biased expression of hsf5-rnf43 in the early development of Nile tilapia, and was affected by high temperature during the thermosensitive period of Nile tilapia sex differentiation. Therefore, we first reported that the fusion gene hsf5-rnf43 was sex-biased expressed in the testis regulated by DNA methylation and affected by high temperature, which may be involved in the maintenance of testis function and sex differentiation of Nile tilapia.

Gametogenesis of pacific salmon. 1. Pink salmon <i>Oncorhynchus gorbuscha</i>

Cited information on gametogenesis of pink salmon Oncorhynchus gorbuscha Walbaum is summarized. Periods of indifferent development and sex differentiation are considered, development of the testes and ovaries is traced, and the fertility formation is analyzed. Regularities of sex reversion, unique for salmonids, and evolutionarily acquired specific features of oogenesis associated with a short life cycle are noted.

Combined effects of rearing temperature regime (thermocycle vs. constant temperature) during early development and thermal treatment on Nile tilapia (Oreochromis niloticus) sex differentiation

In nature, water temperature experiences daily variations known as thermocycles. Temperature is the main environmental factor that influences sex determination in most teleost fish. The purpose of this study was to examine the effects of rearing temperature (thermocycle (TC) vs. constant (CTE)) on development and a posterior thermal shock throughout the period of sex differentiation of Nile tilapia (Oreochromis niloticus). Embryos and larvae were kept under two temperature regimes: TC of 31 °C:25 °C day:night vs. CTE of 28 °C from 0 to 11 dpf. After this period, the larvae from each group were subjected to either heat treatment (HT, 36 °C for 12 days) or kept under the same rearing temperatures until 23 dpf (Control, C). Then all the groups remained at constant temperature until 270 dpf, when blood and gonads were collected. Larval samples were used to examine the expression of genes related to male (amh, ara, sox9a, dmrt1a) and female (cyp19a1a, foxl2, era) sexual differentiation. In juveniles, sex was characterized by histology, the gonadal expression of the genes involved in the sex steroid synthesis was analyzed by qPCR, and plasma testosterone (T) and estradiol (E2) levels were analyzed by ELISA. In larvae, daily TCs increased the survival rate against HT and up-regulated the expression of ovarian differentiation genes. In juveniles, TC + C induced a higher proportion of females and higher cyp19a1a expression compared to CTE + C. HT induced changes in the CTE group by up-regulating testicular differentiation genes and down-regulating female promoting genes, which did not occur in the TC group. Juveniles from TC + C group presented a higher proportion of females with higher E2 and cyp19a1a than CTE + HT. Fish from the CTE + HT group showed a higher percentage of males with highest T and amh. These findings indicate that daily TCs during larval development promote ovarian differentiation and diminish the masculinizing effects of HT.

Microcystin-LR immersion caused sequential endocrine disruption and growth inhibition in zebrafish (Danio rerio) from fertilization to sexual differentiation completion

Microcystin-LR (MC-LR) is a highly toxic congener and is also one of the most commonly found. Recent studies have demonstrated that MC-LR can disrupt growth and endocrine in fish, but how it works at the stage of the sex differentiation period had not been determined to date. In this study, zebrafish (Danio rerio) embryos were exposed to MC-LR (0 and 10 μg/L), and sampled at 14, 28, and 42 days post fertilization (dpf), respectively. The results demonstrated that MC-LR caused the growth inhibition of zebrafish at 42 dpf. The expression levels of genes related to the growth hormone/insulin-like growth factor (GH/IGF) and hypothalamic-pituitary-thyroid (HPT) axes, as well as the levels of hormone 3,5,3′- triiodothyronine (T3) and thyroxine (T4), were significantly decreased at all time points. A significant decrease in the ratio of testosterone and estradiol (T/E2) were detected at 28 and 42 dpf in MC-LR group along with changes in genes related to the hypothalamic-pituitary–gonadal (HPG) axis. The result of sex ratio showed that the percentage of females was up to 61.84%, indicating a estrogenic effect induced by MC-LR. The significant changes on hormone levels and gene transcripts occurred mainly in the stage of sex differentiation. The correlation analysis further suggested that key cross-talks among three endocrine axes may be the growth hormone releasing hormone (GHRH), transthyretin (TTR) and gonadotropin releasing hormone (GnRH) signaling molecules. Overall, our findings provide a new insight for understanding the mechanisms by which MC-LR affects fish growth and reproduction during gonadal development.

Sex Reversal Induced by Dietary Supplementation with 17α-Methyltestosterone during the Critical Period of Sex Differentiation in Oriental River Prawn (Macrobrachium nipponense).

The steroid 17α-methyltestosterone (MT) inhibits ovarian function and is often used to induce sex reversal artificially in vertebrates. In the present study, different concentrations of MT were added as dietary supplementation, and the effects on sex ratio, growth, and gonadal development were examined. After 40 days, the sex ratio (male:female) in each group increased at different degrees with 50 (1.36:1), 100 (1.57:1), and 200 (2.61:1) mg/kg MT, and neo-males with testis-ovary coexistence were observed in the 200 mg/kg MT group. Furthermore, 50 and 100 mg/kg MT could induce female reversion in neo-males. Histologically, the development of the testes in experimental groups was slower, but the ovaries of the experimental and control groups had similar developmental rates. The expression levels of DMRT11E, Foxl2, and SoxE1 in males at 200 mg/kg MT were 8.65-, 3.75-, and 3.45-fold greater than those of the control group. In crustaceans, sex reversal through vertebrate sex hormones can be observed. Neo-males (sex-reversed female prawns) were maintained by exogenous androgen, and over-reliance led to slow testis growth, small body size, and low growth rate, but sperm was still produced. In female prawns, MT inhibited ovary development and promoted growth.

DNA methylation and demethylation shape sexual differentiation of neurochemical phenotype

Some of the best-studied neural sex differences depend on differential cell death in males and females, but other sex differences persist even if cell death is prevented. These include sex differences in neurochemical phenotype (i.e., stable patterns of gene expression). Work in our laboratory over the last several years has tested the hypothesis that sex differences in DNA methylation early in life underlie sexual differentiation of neuronal phenotype. We have shown that 1) expression of enzymes that place or remove DNA methylation marks is greatest during the first week of life in the mouse brain and overlaps with the perinatal critical period of sexual differentiation; 2) a transient inhibition of DNA methylation during neonatal life abolishes several sex differences in cell phenotype in the mouse hypothalamus; 3) both DNA methylation and de-methylation contribute to the development of neural sex differences; and 4) the effects of DNA methylation and de-methylation are brain region- and cell type-specific.

Amh/Amhr2 Signaling Causes Masculinization by Inhibiting Estrogen Synthesis during Gonadal Sex Differentiation in Japanese Flounder (Paralichthys olivaceus).

The anti-Müllerian hormone (Amh) is a protein belonging to the TGF-β superfamily, the function of which has been considered important for male sex differentiation in vertebrates. The Japanese flounder (Paralichthys olivaceus) is a teleost fish that has an XX/XY sex determination system and temperature-dependent sex determination. In this species, amh expression is up-regulated in genetic males and in temperature-induced masculinization during the sex differentiation period. However, to the best of our knowledge, no reports on the Amh receptor (Amhr2) in flounder have been published, and the details of Amh signaling remain unclear. In this study, we produced amhr2-deficient mutants using the CRISPR/Cas9 system and analyzed the gonadal phenotypes and sex-related genes. The results revealed that the gonads of genetically male amhr2 mutants featured typical ovaries, and the sex differentiation-related genes showed a female expression pattern. Thus, the loss of Amhr2 function causes male-to-female sex reversal in Japanese flounder. Moreover, the treatment of genetically male amhr2 mutants with an aromatase inhibitor fadrozole, which inhibits estrogen synthesis, resulted in testicular formation. These results strongly suggest that Amh/Amhr2 signaling causes masculinization by inhibiting estrogen synthesis during gonadal sex differentiation in the flounder.

Comparative transcriptome analysis of early sexual differentiation in the male and female gonads of common carp (Cyprinus carpio)

The growth rate of allotetraploid common carp (Cyprinus carpio) is sex dimorphic. Thus, intervention for controlling sex is crucial for farming this fish. However, few studies have investigated the spatiotemporal interaction of interaction of various genes related to sex determination/early gonadal differentiation in carp. In this study, comparative gonadal transcriptomic analyses were conducted during 4 key sex differentiaton stages for common carp of both sexes including sex determination stage, pre-gonadal, gonadal differentiation, and maturation stages. Altogether, 305.66 Gb raw reads were obtained form 48 libraries. Differentially expressed genes (DEGs) associated with sex differentiation and gonadal development were identified at each time point. Male-specific DEGs including dmrt1, amh, hmga2, sox3, dmrt2, wnt2b, and wnt9b, and female-biased DEGs including foxl2, cyp19a1a, zp3, zp4, and gper1 were identified. The trend analysis of the key genes of sex determination and gonadal differentiation indicated that 35 dph or earlier is the male sex determination period, 90–100 dph is the key window period of male sex differentiation, and 90–100 dph may be the key window period of female sex differentiation. Functional enrichment analysis showed that DEGs at the testicular undifferentiated stages were mainly enriched in androgen receptor signaling, while DEGs from the onset of testicular differentiation to maturity were enriched in male sex differentiation, spermatogenesis, and male gonadal development. In genetic females, DEGs at the undifferentiated ovarian stage are mainly enriched in insulin-like growth factor (IGF) transport, whereas DEGs from the onset of ovarian differentiation to maturation are mainly enriched in ovarian steroidogenesis and oocyte meiosis. Protein-protein interaction (PPI) analysis revealed that 8 DEGs, including Foxl2, Src, Ncor1, and Smad4, were identified as hub proteins. Additionally, plasma 17β-estradiol and 11-Ketotestosterone levels were sexually dimorphic in C. carpo. The study provide insights into the genetic network of the underlying mechanisms of early sex determination and gonadal differentiation in C. carpo for intervening all-female production in better way.

Recent progress of male sexual differentiation and development in the oriental river prawn (Macrobrachium nipponense): A review

AbstractMany researchers have focused on male sexual differentiation and development of the Macrobrachium nipponense in recent years. In this review, we highlight the status of knowledge about male sexual differentiation and development in this species by summarizing the chromosome analyses, major regulatory factors, identification of male reproduction‐related genes and gene functional analyses. Fifty‐two chromosomes have been identified in the testis of M. nipponense, which led to the identification of sex‐related chromosomes in this prawn. Studies have shown that hormones secreted by the eyestalk, androgenic gland, and testis as well as 17‐estradiol and environmental factors regulate the process of male sexual development in M. nipponense. The sex differentiation and development sensitive period was identified as post‐larva 10 (PL10) to PL22; therefore, this time span is critical in the identification of male reproduction‐related genes from M. nipponense. In this study, 80 male sex‐related homologous genes were identified based on functional analysis in other species, and another 45 genes were considered to be strong male reproduction‐related candidates based on their expression changes through performing multi‐omics profiling analyses. Quantitative polymerase chain reaction analysis revealed 25 genes that were involved in the process of male sexual development in M. nipponense, and RNA interference showed that knockdown of the expression levels of SDHB, PDHE1, NFkBa, HSDL1 and ferritin delayed testis development. Use of Crispr‐Cas9 technology and analysis of noncoding RNA should be further investigated, which will be effective in establishing an artificial technique to regulate the process of male sexual development in M. nipponense.

High-temperature stress will put the thermo-sensitive teleost yellow catfish (Tachysurus fulvidraco) in danger through reducing reproductivity

Recently, concerns for species that sex differentiation is influenced by temperature in the context of global warming have increased because disrupted operational sex ratios could threaten population maintenance. In contrast, little attention has been given to the reproductive ability of populations that experienced elevated temperatures. In this study, we demonstrated that high temperature (HT) would decrease population size via three different aspects of reproductive ability for the first time. We show that, in a thermo-sensitive teleost yellow catfish, a short period of HT (+3 °C) exposure during the critical period of sex differentiation leads to a different percentage of masculinization of XX genotypic females (1–23%) in wet-lab and natural water bodies. Combining the results of gonadal appearance, histology, sperm parameters, and fertilization rate, we found that XX pseudo-males induced by HT display significantly discounted fertility and reproductive performance compared to XY normal males. We demonstrate that the survival of the XY genotype is lower than XX genotype under environmental stress, including HT, hypoxia, and parasite infection, and the differential survival seems unrelated to male-biased sexual size dimorphism. The mathematical model predicts that the phenotypic female percent will be stabilized at 50% and the population will be sustainably maintained when masculinizing force is less than 0.5, while HT will put the population in danger when the masculinizing force exceeds 0.5. However, when we combine the real-world data of reproductive ability and mathematic model, our results suggest the population size decreases and the long-term survival of the studied species are threatened under the projected pace of increasing temperature. These findings will be useful for understanding the long-term effects of increasing temperature on sex ratio, reproduction and population maintenance in teleost.

Impact of temperature-induced sex reversal on behavior and sound production in Nile tilapia (Oreochromis niloticus)

In some fish species, sex is determined by the combination of genetic and environmental factors. In most species concerned, extreme temperatures during the sensitive period of sex differentiation drives masculinization, independently of the female sex chromosomes. In Nile tilapia (XY male heterogamety), XX juveniles exposed to high temperatures (>32 °C) can masculinize and become phenotypical males (neomales). Whether these neomales exhibit a different behavior than XY males remains however unclear. Sex reversal being naturally relevant, we investigated the agonistic behavior of neomales during dyadic fights and the preference of gravid females for one of the two male types. We quantified the behavior, size of the nest, hormone circulating levels (testosterone, 11-ketotestosterone and cortisol) and sound production of the two male types in both contexts. Independently of the individual they face, neomales seem to display more aggressive behaviors than XY males but often fail to become dominant. Agonistic interactions were mainly silent, suggesting that sounds are unnecessary for the establishment of social hierarchy. Although males and neomales produce different kinds of sounds when facing a gravid female, the female does not exhibit a preference. Overall, no differences were observed for hormone circulating concentrations between the two male types. We suggest that the sex chromosomes and/or the sex reversal procedure may have differently shaped the brain of neomales, resulting in differences in the expression of behavior.

ESR1 mediates estrogen-induced feminization of genetic male Chinese soft-shelled turtle†.

Exogenous estrogen have shown their feminization abilities during the specific sex differentiation period in several reptiles. However, the specific regulatory mechanism and downstream regulatory genes of estrogen remain elusive. In the present study, 17β-estradiol (E2), as well as drugs of specific antagonists and/or agonists of estrogen receptors, were employed to figure out the molecular pathway involved in the E2-induced feminization in Chinese soft-shelled turtles, an important aquaculture species in China. E2 treatment led to typical female characteristics in the gonads of ZZ individuals, including thickened outer cortex containing a number of germ cells and degenerated medullary cords, as well as the disappearance of male marker SOX9, and the ectopic expression of ovarian regulator FOXL2 at the embryonic developmental stage 27 and 1month after hatching. The specific ESR1 antagonist or a combination of three estrogen receptor antagonists could block the sex reversal of ZZ individuals induced by estrogen. In addition, specific activation of ESR1 by agonist also led to the feminization of ZZ gonads, which was similar to the effect of estrogen treatment. Furthermore, transcriptome data showed that the expression level of FOXL2 was significantly upregulated, whereas mRNA levels of DMRT1, SOX9, and AMH were downregulated after estrogen treatment. Taken together, our results indicated that E2 induced the feminization of ZZ Chinese soft-shelled turtles via ESR1, and decrease of male genes DMRT1, SOX9, and AMH and increase of ovarian development regulator FOXL2 might be responsible for the initiation of E2-induced feminization.

Особливості розвитку репродуктивної системи щурів нащадків чоловічої статі, що народжені від батька, який до спаровування отримував фітоестрогени

The purpose of the study was the research of the influence of rats father's excessive consumption of phytoestrogens on somato-sexual development and reproductive function of male offspring. Materials and methods. Male parents were fed with a mixture of phytoestrogens before mating with intact females at a dose of 20 mg/kg of body weight for 30 days. In reproductive animals, reproductive function was studied for such characteristics as sexual behavior, fertility, spermogram, the mass of androgen-dependent organs was determined, and the level of sex hormones was determined. Experimental parents were divided into two groups: 1) intact, 2) males who received excess phytoestrogens. After the birth of male offspring from these parents, their reproductive system was examined. Results and discussion. Prolonged administration of phytoestrogens to mature male parents (parents) causes absolute and relative hyperestrogenism (five-fold increase in E2 and vice versa reduction of T/E2 three-fold), which leads to a decrease in copulatory activity (reduction of the number of nurseries and intromissions by 28%), violation of spermatogenesis (reduction of sperm concentration by 39%, doubling the proportion of abnormal forms), at the same time does not affect the reproductive potential of male who were born to fathers who received an excess consumption of phytoestrogens. The presence of excess compounds with estrogenic activity in the diet of adult males in the preconsumption period leads to the birth of male offspring, which have reduced testosterone levels (25%) in the critical period of sexual differentiation of the brain (5 days of life). Descendants of the male sex of the phytoestrogenized father have a normal course of somatic development but are characterized by impaired sexual development (less anogenital distance by 13%) and delayed lowering of the testicles into the scrotum, lower body weight in adulthood (28.2% in 150 day rats). Reproductive function of the offspring of phytoestrogenized father is characterized by differences in sexual behavior: less copulatory activity – 50%, less number intromission, the number of males with ejaculation is 10%; delayed sexual reactions; and lower reproductive potential (two-fold reduction of the integrated indicator of potential fertility of animals). Decreased reproductive potential of the offspring, who were born to fathers who received an excess consumption of phytoestrogens, occurs due to a decrease in the proportion of effective fertilization (pregnant females among those fertilized only 60%), indicating negative changes in sperm, the development of which, starting from the stage of spermatocytes, occurred in conditions of absolute and relative hyperestrogenism. Conclusion. The male who were born to fathers who received an excess consumption of phytoestrogens, have complications in the functioning of the reproductive system

Development and gene expression analysis of gonad during 17α-methyltestosterone-induced sex reversal in mandarin fish (Siniperca chuatsi)

Mandarin fish (Siniperca chuatsi) is one of the most important commercial fish in aquaculture in the world. As females grow faster than males, the cultivation of all-female mandarin fish has great economic value and commercial prospects. However, little is known about the molecular mechanism of the early gonad development and sex reversal of mandarin fish. In this study, histological methods and transcriptome sequencing techniques were used to study the early gonadal development and gene expression pattern of genetically female (XX) mandarin fish during MT-induced masculinization. The histological results showed that the labile period of sex differentiation of XX mandarin fish was before 36 dpf and MT treatment before this period could induce complete sex reversal. The transcriptome results showed that the expression levels of a few female-related genes were down-regulated by MT during the morphological differentiation stage to cytological differentiation stage of XX gonad, such as cyp19a1a, foxl2, foxl3, hsd17b1, figla, gdf9, bmp15 and β-catenin, suggesting that the changes of these genes may determine the direction of sex differentiation in mandarin fish. In addition, MT also activated the expression of some male-related genes, such as dmrt1, hsd17b3, hsd11b2, cyp11a1, cyp11b, amh, amhr2, sox9 and gsdf. Some of these genes were activated during spermatogenesis stage, suggesting that the up-regulation of these male-related genes may be the result of sex differentiation rather than the cause. The qRT-PCR results of eight randomly selected differentially expressed genes were basically consistent with the transcriptome results, which proved that the transcriptome data were reliable. These results indicated that the expression of genes related to estrogen synthesis in early development stage may play a decisive role in the direction of sex differentiation in mandarin fish.

A Multidimensional Matrix Model for Predicting the Effects of Male-Biased Sex Ratios on Fish Populations.

Laboratory experiments have established that exposure to certain endocrine-active substances prior to and/or during the period of sexual differentiation can lead to skewed sex ratios in fish. However, the potential long-term population impact of biased sex ratio depends on multiple factors including the life history of the species and whether the ratio is male or female-biased. In the present study, we describe a novel multidimensional, density-dependent matrix model that analyzes age class-structure of both males and females over time, allowing for the quantitative evaluation of the effects of biased sex ratio on population status. This approach can be used in conjunction with field monitoring efforts and/or laboratory testing to link effects on sex ratio due to chemical and/or nonchemical stressors to adverse outcomes in whole organisms and populations. For demonstration purposes, we applied the model to evaluate population trajectories for fathead minnow (Pimephales promelas) exposed to prochloraz, an aromatase inhibitor, during sexual differentiation. The model also was used to explore the population impact in a more realistic exposure scenario in which both adult and early life stages of fish are exposed concurrently to prochloraz, which, in addition to altering sex ratio during development, can decrease vitellogenin and egg production in adult females. For each exposure scenario, the model was used to analyze total population size, numbers of females and of males, and sex specific recruitment of the F1 generation. The present study illustrates the utility of multidimensional matrix population models for ecological risk assessment in terms of integrating effects across a population of an organism even when chemical effects on individuals are manifested via different pathways depending on life stage. Environ Toxicol Chem 2022;41:1066-1077. Published 2022. This article is a U.S. Government work and is in the public domain in the USA.

Gonadal development and molecular analysis revealed the critical window for sex differentiation, and E2 reversibility of XY-male spotted scat, Scatophagus argus

In vertebrate the development of gonad into testis or ovary requires the combined effect of genes and hormones to trigger differentiation from a bipotential gonadal primordium. Knowledge of the gonad development process is vital in studies on fish sex determination and differentiation. The spotted scat (Scatophagus argus) is an economically important marine fish with an XX/XY sex-determining system and exhibits sexual growth dimorphism in favor of females. Meanwhile, information on gonad development and gene expression patterns in the early gonads is unknown. This study explores the pattern of spotted scat early gonad development by morphological and molecular means. In the undifferentiated gonads, the number of germ cells in females is not significantly different from males. At age 3 mph (months post-hatch), the number of germ cells in females was significantly higher than in males. The sex differentiation began in females before males with the proliferation of primary oocyte and spermatocytes at 3.5 and 4 mph, respectively. In the early gonads, dmrt1 and amh were significantly higher in XY-fish, while cyp19a1a and foxl2 were higher in the XX-fish. Within the critical period of sex differentiation, estradiol treatment was able to sex-reverse XY-male to XY-female. In the sex-reversed group, male (dmrt1 and amh) and female (cyp19a1a and foxl2) related genes were suppressed and increased, respectively. 17α-Methyltestosterone or aromatase inhibitor (letrozole) could not induce sex reversal in XX-fish due to the absence of the male-specific dmrt1 required for testes development. Conclusively, this study identifies the critical window for sex differentiation and significantly higher germ cell numbers in future females than males. We provide evidence that the phenotypic XY-male is reversible, while the phenotypic XX-female is irreversible. Dmrt1 is necessary for testis differentiation, amh in males, and cyp19a1a and foxl2 in females could also be used as molecular markers in early gonads. This study explicitly presents valuable information for research development on sex determination and differentiation and sex control in spotted scat.

Effects of Diethylstilbestrol on Zebrafish Gonad Development and Endocrine Disruption Mechanism.

Environmental estrogen is a substance that functions as an endocrine hormone in organisms and can cause endocrine system disruption. A typical environmental estrogen, diethylstilbestrol (DES), can affect normal sexual function and organism development. However, even though the effects of different exposure stages of DES on the endocrine system and gonadal development of zebrafish juveniles are unknown, sex determination is strongly influenced by endocrine-disrupting chemicals (EDCs). From 10–90 days post fertilization (dpf), juvenile zebrafish were exposed to DES (100 and 1000 ng/L) in three different stages (initial development stage (IDS), 10–25 dpf; gonadal differentiation stage (GDS), 25–45 dpf and gonadal maturity stage (GMS), 45–60 dpf). Compared with that of IDS and GMS, the growth indicators (body length, body weight, and others) decreased significantly at GDS, and the proportion of zebrafish females exposed to 100 ng/L DES was significantly higher (by 59.65%) than that of the control; in addition, the zebrafish were biased towards female differentiation. The GDS is a critical period for sex differentiation. Our results show that exposure to environmental estrogen during the critical gonadal differentiation period not only affects the development of zebrafish, but also affects the population development.