Estrogens regulate early embryonic development of the olfactory sensory system via estrogen-responsive glia

ABSTRACT Estrogens are well-known to regulate development of sexual dimorphism of the brain; however, their role in embryonic brain development prior to sex-differentiation is unclear. Using estrogen biosensor zebrafish models, we found that estrogen activity in the embryonic brain occurs from early neurogenesis specifically in a type of glia in the olfactory bulb (OB), which we name estrogen-responsive olfactory bulb (EROB) cells. In response to estrogen, EROB cells overlay the outermost layer of the OB and interact tightly with olfactory sensory neurons at the olfactory glomeruli. Inhibiting estrogen activity using an estrogen receptor antagonist, ICI182,780 (ICI), and/or EROB cell ablation impedes olfactory glomerular development, including the topological organisation of olfactory glomeruli and inhibitory synaptogenesis in the OB. Furthermore, activation of estrogen signalling inhibits both intrinsic and olfaction-dependent neuronal activity in the OB, whereas ICI or EROB cell ablation results in the opposite effect on neuronal excitability. Altering the estrogen signalling disrupts olfaction-mediated behaviour in later larval stage. We propose that estrogens act on glia to regulate development of OB circuits, thereby modulating the local excitability in the OB and olfaction-mediated behaviour.

Deciphering sex-specific miRNAs as heat-recorders in zebrafish

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression in a wide variety of physiological processes, including those related to the reproductive system. Although in the last decade a plethora of miRNAs has been reported, the miRNA alterations occurred by environmental cues and their biological functions have not yet been elucidated. With the aim to identify epigenetic regulations mediated by miRNAs in the gonads in a climate change scenario, zebrafish (Danio rerio) were subjected to high temperatures during sex differentiation (18-32 days post fertilization, dpf), a treatment that results in male-skewed sex ratios. Once the fish reached adulthood (90 dpf), ovaries and testes were sequenced by high-throughput technologies. About 101 million high-quality reads were obtained from gonadal samples. Analyses of the expression levels of the miRNAs identified a total of 23 and 1 differentially expressed (DE) miRNAs in ovaries and testes, respectively, two months after the heat treatment. Most of the identified miRNAs were involved in human sex-related cancer. After retrieving 3’ UTR regions, ~400 predicted targets of the 24 DE miRNAs were obtained, some with reproduction-related functions. Their synteny in the zebrafish genome was, for more than half of them, in the chromosomes 7, 2, 4, 3 and 11 in the ovaries, chromosome 4 being the place where the predicted sex-associated-region (sar) is localized in wild zebrafish. Further, spatial localization in the gonads of two selected miRNAs (miR-122-5p and miR-146-5p) showed exclusive expression in the ovarian germ cells. The present study expands the catalog of sex-specific miRNAs and deciphers, for the first time, thermosensitive miRNAs in the zebrafish gonads that might be used as potential epimarkers to predict environmental past events.

Genes in the Azoospermia Factor A Region of the Y Chromosome Show Sexual Dimorphism in Rat Brain Prior to Gonadal Sex Differentiation

BackgroundThe classical concept of brain sex differentiation suggests that steroid hormones released from the gonads program male and female brains differently. However, several studies indicate that steroid hormones are not the only determinant of brain sex differentiation and that genetic differences could also be involved.MethodsIn this study, we have performed RNA sequencing of rat brains at embryonic days 12 (E12), E13, and E14. The aim was to identify differentially expressed genes between male and female rat brains during early development. ResultsAnalysis of genes expressed with the highest sex differences showed that Xist was highly expressed in females having XX genotype with an increasing ratio over time. Analysis of genes expressed with the highest male expression identified three main genes. At E12, two genes located in the azoospermia factor A (AZFa) region on the Y chromosome were highly expressed in males. These were Ddx3y (1552-fold higher in males) and Kdm6c (147-fold higher in males). The expression of Kdm6c, but not Ddx3y, remained high at both E13 and E14. In qRT-PCR analysis, these two genes were highly expressed in all the stages in male brain. In addition to these genes, one of the several copies of Sry in the rat genome, Sry4, showed a high expression in the male brains at all three time points. At all three time points several other genes were also found to show sex bias, but with lower differences in gene expression. ConclusionThe observed sex-specific expression of genes at early development suggests that the rat brain is sexually dimorphic prior to gonadal action on the brain and identifies the AZFa region genes as a possible contributor to male brain development.

A spatiotemporal steroidogenic regulatory network in human fetal adrenal glands and gonads

Human fetal adrenal glands produce substantial amounts of dehydroepiandrosterone (DHEA), which is one of the most important precursors of sex hormones. However, the underlying biological mechanism remains largely unknown. Herein, we sequenced human fetal adrenal glands and gonads from 7 to 14 GW via the 10× Genomics single-cell transcriptome techniques and reconstructed their location information by Spatial Transcriptome, conducted COOL-seq for the MC2R+ inner zone steroidogenic cells during the time window of sex differentiation (8-12GW). We found that relative to gonads, adrenal glands begin to synthesize steroids early. The coordination among steroidogenic cells and multiple nonsteroidogenic cells promotes adrenal cortex construction and steroid synthesis. Notably, during the time window of sex differentiation (8-12 GW), key enzyme gene expression shifts to accelerate DHEA synthesis in males and cortisol synthesis in females. Furthermore, high SST+ expressions in the adrenal gland and testis amplify androgen synthesis in males. Our research highlights the robustness of the action of fetal adrenal glands on gonads to modify the process of sexual differentiation.

Genetic Control of MAP3K1 in Eye Development and Sex Differentiation

The MAP3K1 is responsible for transmitting signals to activate specific MAP2K-MAPK cascades. Following the initial biochemical characterization, genetic mouse models have taken center stage to elucidate how MAP3K1 regulates biological functions. To that end, mice were generated with the ablation of the entire Map3k1 gene, the kinase domain coding sequences, or ubiquitin ligase domain mutations. Analyses of the mutants identify diverse roles that MAP3K1 plays in embryonic survival, maturation of T/B cells, and development of sensory organs, including eye and ear. Specifically in eye development, Map3k1 loss-of-function was found to be autosomal recessive for congenital eye abnormalities, but became autosomal dominant in combination with Jnk and RhoA mutations. Additionally, Map3k1 mutation increased eye defects with an exposure to environmental agents such as dioxin. Data from eye developmental models reveal the nexus role of MAP3K1 in integrating genetic and environmental signals to control developmental activities. Here, we focus the discussions on recent advances in understanding the signaling mechanisms of MAP3K1 in eye development in mice and in sex differentiation from human genomics findings. The research works featured here lead to a deeper understanding of the in vivo signaling network, the mechanisms of gene–environment interactions, and the relevance of this multifaceted protein kinase in disease etiology and pathogenesis.

Towards accurate and simple morphometric sex differentiation in Bonelli’s Eagle Aquila fasciata nestlings: Interpopulation variations and influence of growth conditions

Morphometric methods of sex differentiation may be cheap, simple, quick, and reliable alternatives to molecular approaches. However, there are still important uncertainties regarding the use of morphometric methods in birds, particularly regarding their applicability to different populations and environmental conditions. Between 2004 and 2019, we sampled 245 Bonelli´s Eagle Aquila fasciata nestlings, 197 from nests in Catalonia (northeastern Spain) and Andalusia (southeastern Spain) and 56 that were partially or totally raised in captivity. Our objective was to develop morphometry-based sexing methods for Bonelli’s Eagle nestlings that can be applied in situ to different subpopulations and growth conditions. We recorded up to nine measures related to nestling body mass, bill, tarsus, and claw length. Tail and wing length were used to control for the age of nestlings, and all individuals were sexed genetically. We found important morphometric differences between the two natural subpopulations. According to our discriminant analyses, body mass and claw length were the most discriminant variables, both in wild and captivity conditions. Differences between sexes were more prominent during the flight feathers’ growing period. Feeding ad libitum in captivity conditions led to larger nestlings and reduced between-sexes differences. We provide different models for sexing Bonelli’s Eagle nestlings that can be applied according to the studied geographic location, growth conditions (wild versus captive), and balance between accuracy and simplicity. Overall, our findings highlight that extrapolating sex discriminant functions to different subpopulations and growth conditions may be risky.

The Patterns of Male and Female Flowers in Flowering Stage May Not Be Optimal Resource Allocation for Fruit and Seed Growth

Changes in the proportions of male and female flowers in monoecious plants in response to external environmental conditions are directly related to the reproductive fitness of plants. The monoecious cucumber (Cucumber sativus) plant was used in this study to assess the responses of sex differentiation and the breeding process to nutrient supply and the degree of artificial pollination using pollen solutions of different concentrations. We found that the nutrient supply significantly improved the number of female flowers, while pollination treatments did not obviously increase the number of male flowers. Continuous pollination changed the number of female flowers especially in the later stage of the pollination experiment. Therefore, pollination changed the ratio of male and female flowers in the flowering stage of cucumber. Pollination treatment affected the fruit growth, seed set, and fruit yield. The number of fruit, fruit set percentage, and total seeds per plant did not increase with the pollination level, but individual fruit weight and seed number in one fruit did increase. The differentiation of male and female flowers in the flowering stage of cucumber is a response to nutrient and pollination resources, but this response is not the optimal resource allocation for subsequent fruit development and seed maturity, which suggests that the response of plants to external environment resources is short-term and direct.

Intragenic Duplication of DMRT1 in a SRY-Negative Boy with 46,XX Disorder of Sex Development

Background. 46,XX disorders of sex development are rare. Approximately, 90% of XX males are SRY-positive, while testicular development in the absence of SRY takes place in a minority. Methods: A boy with 46,XX karyotype (SRY-negative; absence of SOX9 duplications) was investigated by targeted Next Generation Sequencing (NGS), Multiplex ligation-dependent probe amplification (MLPA), and Comparative Genomic Hybridization array (CGH-array). Results: The boy had normal male phenotype and normal prepubertal values of testicular hormones. He presented a heterozygous duplication of 49.626 bp, encompassing exons 2 and 3 of DMRT1. The result was arr[GRCh37] 9p24.3(845893_895518)x3. Since both breakpoints are harbored in the intronic regions, the duplication does not stop or shift the coding frame. Additional known pathogenic or uncertain variants in pro-testis gene cascade were not identified. Conclusions: This study report a boy with 46,XX testicular disorder of sex differentiation, showing a de novo partial intragenic duplication of DMRT1. This intragenic duplication may result in a gain of function, acting as primary pro-testis gene (or anti-ovary gene) in a 46,XX human foetus and permitting normal pre-pubertal endocrine testis function.

Comparative Transcriptome Analysis of Female and Male Fine-Patterned Puffer: Identification of Candidate Genes Associated with Growth and Sex Differentiation

Sexual growth dimorphism is a common phenomenon in teleost fish. However, the mechanism of this complex phenomenon remains unclear. The fine-patterned puffer (Takifugu poecilonotus; Temminck and Schlegel, 1850) exhibits female-biased sexual size dimorphism similar to other pufferfish. In this study, the transcriptomes of female and male T. poecilonotus were sequenced, 285.95 million raw read pairs were generated from sequence libraries. After identification and assembly, a total of 149,814 nonredundant unigenes were obtained with an N50 length of 3538 bp. Of these candidates, 122,719 unigenes (81.91% of the total) were successfully annotated with multiple public databases. The comparison analysis revealed 10,385 unigenes (2034 in females and 8351 in males) were differentially expressed between different sexes of T. poecilonotus. Then, we identified many candidate growth- and sex-related genes, including Dmrt1, Sox3, Spatas, Prl/Prlr, fabps, Ghr, and Igf1r. In addition to these well-known genes, Fabp4 was identified for the first time in fish. Furthermore, 68,281 simple sequence repeats (SSRs) loci were screened and identified in the transcriptome sequence of T. poecilonotus. The results of our study could provide valuable information on growth- and sex-associated genes and facilitate further exploration of the molecular mechanism of sexual growth dimorphism.