The Important Role of Sex-Related Sox Family Genes in the Sex Reversal of the Chinese Soft-Shelled Turtle (Pelodiscus sinensis)

The Chinese soft-shelled turtle Pelodiscus sinensis shows obvious sexual dimorphism. The economic and nutrition value of male individuals are significantly higher than those of female individuals. Pseudo-females which are base to all-male breeding have been obtained by estrogen induction, while the gene function and molecular mechanism of sex reversal remain unclear in P. sinensis. Here, comparative transcriptome analyses of female, male, and pseudo-female gonads were performed, and 14,430 genes differentially expressed were identified in the pairwise comparison of three groups. GO and KEGG analyses were performed on the differentially expressed genes (DEGs), which mainly concentrated on steroid hormone synthesis. Furthermore, the results of gonadal transcriptome analysis revealed that 10 sex-related sox genes were differentially expressed in males vs. female, male vs. pseudo-female, and female vs. pseudo-female. Through the differential expression analysis of these 10 sox genes in mature gonads, six sox genes related to sex reversal were further screened. The molecular mechanism of the six sox genes in the embryo were analyzed during sex reversal after E2 treatment. In mature gonads, some sox family genes, such as sox9sox12, and sox30 were highly expressed in the testis, while sox1, sox3, sox6, sox11, and sox17 were lowly expressed. In the male embryos, exogenous estrogen can activate the expression of sox3 and inhibit the expression of sox8, sox9, and sox11. In summary, sox3 may have a role in the process of sex reversal from male to pseudo-female, when sox8 and sox9 are inhibited. Sox family genes affect both female and male pathways in the process of sex reversal, which provides a new insight for the all-male breeding of the Chinese soft-shelled turtle.

Roles of Anti-Müllerian hormone (Amh) and its duplicates in sex determination and germ cell proliferation of Nile tilapia

Duplicates of amh are crucial for fish sex determination and differentiation. In Nile tilapia, unlike in other teleosts, amh is located on X chromosome. The Y chromosome amh (amh△-y) is mutated with 5 bp insertion and 233 bp deletion in the coding sequence, and tandem duplicate of amh on Y chromosome (amhy) has been identified as the sex determiner. However, the expression of amh, amh△-y and amhy, their roles in germ cell proliferation and the molecular mechanism of how amhy determines sex is still unclear. In this study, expression and functions of each duplicate were analyzed. Sex reversal occurred only when amhy was mutated as revealed by single, double and triple mutation of the three duplicates in XY fish. Homozygous mutation of amhy in YY fish also resulted in sex reversal. Earlier and higher expression of amhy/Amhy was observed in XY gonads compared with amh/Amh during sex determination. Amhy could inhibit the transcription of cyp19a1a through Amhr2/Smads signaling. Loss of cyp19a1a rescued the sex reversal phenotype in XY fish with amhy mutation. Interestingly, mutation of both amh and amhy in XY fish or homozygous mutation of amhy in YY fish resulted in infertile females with significantly increased germ cell proliferation. Taken together, these results indicated that up-regulation of amhy during the critical period of sex determination makes it the sex-determining gene, and it functions through repressing cyp19a1a expression via Amhr2/Smads signaling pathway. Amh retained its function in controlling germ cell proliferation as reported in other teleosts, while amh△-y was nonfunctionalized.

Sexual Development Dysgenesis in Interspecific Hybrids of Rice 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. Our results provide molecular clues for the proximate mechanisms of hybrid incompatibility and Haldane’s rule.

Dynamics of epigenetic modifiers and environmentally sensitive proteins in a reptile with temperature induced sex reversal

The mechanisms by which sex is determined, and how a sexual phenotype is stably maintained during adulthood, has been the focus of vigorous scientific inquiry. Resources common to the biomedical field (automated staining and imaging platforms) were leveraged to provide the first immunofluorescent data for a reptile species with temperature induced sex reversal. Two four-plex immunofluorescent panels were explored across three sex classes (sex reversed ZZf females, normal ZWf females, and normal ZZm males). One panel was stained for chromatin remodelling genes JARID2 and KDM6B, and methylation marks H3K27me3, and H3K4me3 (Jumonji Panel). The other CaRe panel stained for environmental response genes CIRBP and RelA, and H3K27me3 and H3K4me3. Our study characterised tissue specific expression and cellular localisation patterns of these proteins and histone marks, providing new insights to the molecular characteristics of adult gonads in a dragon lizard Pogona vitticeps. The confirmation that mammalian antibodies cross react in P. vitticeps paves the way for experiments that can take advantage of this new immunohistochemical resource to gain a new understanding of the role of these proteins during embryonic development, and most importantly for P. vitticeps, the molecular underpinnings of sex reversal.

Sex Reversal and Performance in Fitness-Related Traits During Early Life in Agile Frogs

Sex reversal is a mismatch between genetic sex (sex chromosomes) and phenotypic sex (reproductive organs and secondary sexual traits). It can be induced in various ectothermic vertebrates by environmental perturbations, such as extreme temperatures or chemical pollution, experienced during embryonic or larval development. Theoretical studies and recent empirical evidence suggest that sex reversal may be widespread in nature and may impact individual fitness and population dynamics. So far, however, little is known about the performance of sex-reversed individuals in fitness-related traits compared to conspecifics whose phenotypic sex is concordant with their genetic sex. Using a novel molecular marker set for diagnosing genetic sex in agile frogs (Rana dalmatina), we investigated fitness-related traits in larvae and juveniles that underwent spontaneous female-to-male sex reversal in the laboratory. We found only a few differences in early life growth, development, and larval behavior between sex-reversed and sex-concordant individuals, and altogether these differences did not clearly support either higher or lower fitness prospects for sex-reversed individuals. Putting these results together with earlier findings suggesting that sex reversal triggered by heat stress may be associated with low fitness in agile frogs, we propose the hypothesis that the fitness consequences of sex reversal may depend on its etiology.

Effects of synthetic androgen (17α-methyltestosterone) and estrogen (17β-estradiol) on growth and skin coloration in emperor red cichlid, Aulonocara nyassae (Actinopterygii: Cichliformes: Cichlidae)

In recent years, the use of anabolic steroids in the coloration and growth of fishes, especially ornamental ones, has attracted great interest. In the ornamental fish industry, it is economically advantageous to produce some species with high commercial value and higher demand, depending on size, color, and sex. Therefore, the most commonly used steroids in this study—i.e., 17α-MT and 17β-Es (E2)—were added to the diet of emperor red cichlid, Aulonocara nyassae Regan, 1922, which has not been previously hormone-treated and has high economic value amongst ornamental fishes. A 60-day study was conducted in a closed system, where the juveniles of the emperor red cichlid were acclimatized with the control/basal diet for 15 days. After which, 15 fish with a similar shade of color and about 5 months old were weighed individually (0.71 ± 0.01 g). All fish were placed into aquaria (30 L) in five different groups, in triplicate. Five different groups consisted of control (without hormone), 50 mg · kg–1 17α-MT, 100 mg · kg–1 17α-MT, 50 mg · kg–1 E2, and 100 mg · kg–1 E2. The fish were fed a diet twice a day (10:00 h, 17:00 h) for 60 days till satiation. During the entire trial period, a 12 h light–12 h dark photoperiod was maintained. Water temperature was measured daily and recorded. Growth parameters of experimental fish were calculated. The color measurement of fish skin (L*, a*, b* values) from around the dorsal section was performed using a colorimeter (Konica Minolta CR 400). Significant differences were determined in the following parameters: weight gain (WG), specific growth rate (SGR), feed conversion rate (FCR), survival rate (SR), condition factor (CF), and sex reversal. The fish group fed with 17α-MT displayed brighter coloration as compared to other groups. Color analysis (instrumental) in terms of L*, a*, and b*, values showed that the group fed with 17α-MT displayed brighter coloration compared to other groups (P < 0.05). In terms of sex reversal, the fish in the 17α-MT groups exhibited 100% masculinization, whereas in E2 supplemented fish groups (50 and 100 mg · kg–1), the feminization rates were 88.88% and 93.33%, respectively. In conclusion, both hormones were found to have positive and negative effects for this fish species, but the 17α-MT hormone was found to be more effective in reversing skin pigmentation, growth, and sexing, which is the main driver in the ornamental fish trade.

Sex-Biased Mortality and Sex Reversal Shape Wild Frog Sex Ratios

Population sex ratio is a key demographic factor that influences population dynamics and persistence. Sex ratios can vary across ontogeny from embryogenesis to death and yet the conditions that shape changes in sex ratio across ontogeny are poorly understood. Here, we address this issue in amphibians, a clade for which sex ratios are generally understudied in wild populations. Ontogenetic sex ratio variation in amphibians is additionally complicated by the ability of individual tadpoles to develop a phenotypic (gonadal) sex opposite their genotypic sex. Because of sex reversal, the genotypic and phenotypic sex ratios of entire cohorts and populations may also contrast. Understanding proximate mechanisms underlying phenotypic sex ratio variation in amphibians is important given the role they play in population biology research and as model species in eco-toxicological research addressing toxicant impacts on sex ratios. While researchers have presumed that departures from a 50:50 sex ratio are due to sex reversal, sex-biased mortality is an alternative explanation that deserves consideration. Here, we use a molecular sexing approach to track genotypic sex ratio changes from egg mass to metamorphosis in two independent green frog (Rana clamitans) populations by assessing the genotypic sex ratios of multiple developmental stages at each breeding pond. Our findings imply that genotypic sex-biased mortality during tadpole development affects phenotypic sex ratio variation at metamorphosis. We also identified sex reversal in metamorphosing cohorts. However, sex reversal plays a relatively minor and inconsistent role in shaping phenotypic sex ratios across the populations we studied. Although we found that sex-biased mortality influences sex ratios within a population, our study cannot say at this time whether sex-biased mortality is responsible for sex ratio variation across populations. Our results illustrate how multiple processes shape sex ratio variation in wild populations and the value of testing assumptions underlying how we understand sex in wild animal populations.

Masculinization of betta fish (Betta sp) using cow testic flour through Artemia Immersion

Betta fish (Betta sp) is a freshwater ornamental fish during the pandemic that is very popular with the public. However, people tend to prefer male betta fish than female fish. To deal with the large number of requests, a sex reversal technique is carried out which applies hormonal engineering to change female to male sexual characters (masculinization) using natural ingredients, namely cow testicle flour. The purpose of this study was to analyze the dose of bovine testicular flour hormone on the process of oral male betta fish larvae. This research was conducted in Moncongloe Village, Maros Regency, from July to September 2021. The production of beef testicle flour was carried out in the Chemical Engineering laboratory, Ujung Pandang State Polytechnic. Betta fish seeds were kept each in an aquarium measuring 20 cm x 14 cm x 20 cm as many as 12 pieces. Soaking artemia with beef testicle flour with a predetermined dose of 60 mg L-1, 80 mg L-1 and 100 mg L-1, the duration of immersion is 24 hours. This study used a one-factor completely randomized design (CRD). The results obtained were the highest percentage of male genitalia at a dose of 100 mg L-1 with a percentage of 66.7%. The survival rate of betta fish during the maintenance period with different doses of cow testicles obtained data ranging from 80-100%.