The homeobox gene TGIF1 is required for chicken ovarian cortical development and generation of the juxtacortical medulla.

During early embryogenesis in amniotic vertebrates, the gonads differentiate into either ovaries or testes. The first cell lineage to differentiate gives rise to the supporting cells: Sertoli cells in males and pre-granulosa cells in females. These key cell types direct the differentiation of the other cell types in the gonad, including steroidogenic cells. The gonadal surface epithelium and the interstitial cell populations are less well studied, and little is known about their sexual differentiation programs. Here, we show the requirement of the homeobox transcription factor gene TGIF1 for ovarian development in the chicken embryo. TGIF1 is expressed in the two principal ovarian somatic cell populations: the cortex and the pre-granulosa cells of the medulla. TGIF1 expression is associated with an ovarian phenotype in estrogen-mediated sex reversal experiments. Targeted misexpression and gene knockdown indicate that TGIF1 is required, but not sufficient, for proper ovarian cortex formation. In addition, TGIF1 is identified as the first known regulator of juxtacortical medulla development. These findings provide new insights into chicken ovarian differentiation and development, specifically cortical and juxtacortical medulla formation.

Diaph1 knockout inhibits mouse primordial germ cell proliferation and affects gonadal development.

BACKGROUND: Exploring the molecular mechanisms of primordial germ cell (PGC) migration and the involvement of gonadal somatic cells in gonad development is valuable for comprehending the origins and potential treatments of reproductive-related diseases. METHODS: Diaphanous related formin 1 (Diaph1, also known as mDia1) was screened by analyzing publicly available datasets (ATAC-seq, DNase-seq, and RNA-seq). Subsequently, the CRISPR-Cas9 technology was used to construct Diaph1 knockout mice to investigate the role of Diaph1 in gonad development. RESULTS: Based on data from public databases, a differentially expressed gene Diaph1, was identified in the migration of mouse PGC. Additionally, the number of PGCs was significantly reduced in Diaph1 knockout mice compared to wild type mice, and the expression levels of genes related to proliferation (Dicer1, Mcm9), adhesion (E-cadherin, Cdh1), and migration (Cxcr4, Hmgcr, Dazl) were significantly decreased. Diaph1 knockout also inhibited Leydig cell proliferation and induced apoptosis in the testis, as well as granulosa cell apoptosis in the ovary. Moreover, the sperm count in the epididymal region and the count of ovarian follicles were significantly reduced in Diaph1 knockout mice, resulting in decreased fertility, concomitant with lowered levels of serum testosterone and estradiol. Further research found that in Diaph1 knockout mice, the key enzymes involved in testosterone synthesis (CYP11A1, 3ß-HSD) were decreased in Leydig cells, and the estradiol-associated factor (FSH receptor, AMH) in granulosa cells were also downregulated. CONCLUSIONS: Overall, our findings indicate that the knockout of Diaph1 can disrupt the expression of factors that regulate sex hormone production, leading to impaired secretion of sex hormones, ultimately resulting in damage to reproductive function. These results provide a new perspective on the molecular mechanisms underlying PGC migration and gonadal development, and offer valuable insights for further research on the causes, diagnosis, and treatment of related diseases.

The expression profiles of cyp19a1, sf-1, esrs and gths in the brain-pituitary during gonadal sex differentiation in juvenile Japanese eels.

Eels are gonochoristic species whose gonadal differentiation initiates at the yellow eel stage and is influenced by environmental factors. We revealed some sex-related genes were sex dimorphically expressed in gonads during gonadal sex differentiation of Japanese eel (Anguilla japonica); however, the expression of sex-related genes in the brain-pituitary during gonadal sex differentiation in eels is still unclear. This study aimed to investigate the sex-related gene expressions in the brain-pituitary and tried to clarify their roles in the brain and gonads during gonadal sex differentiation. Based on our previous histological study, the control eels developed as males, and estradiol-17ß (E2) was used for feminization. Our results showed that during testicular differentiation, the brain cyp19a1 transcripts and aromatase proteins were increased significantly; moreover, the cyp19a1, sf-1, foxl2s, and esrs (except gperb) transcripts in the midbrain/pituitary also were increased significantly. Forebrain gnrh1 transcripts increased slightly during gonadal differentiation of both sexes, but the gnrhr1b and gnrhr2 transcripts in the midbrain/pituitary were stable during gonadal differentiation. The expression levels of gths and gh in the midbrain/pituitary were significantly increased during testicular differentiation and were much higher in males than in E2-feminized females. These results implied that endogenous estrogens might play essential roles in the brain/pituitary during testicular differentiation, sf-1, foxl2s, and esrs may have roles in cyp19a1 regulation in the midbrain/pituitary of Japanese eels. For the GnRH-GTH axis, gths, especially fshb, may be regulated by esrs and involved in regulating testicular differentiation and development in Japanese eels.

Primary sex determination in birds depends on DMRT1 dosage, but gonadal sex does not determine adult secondary sex characteristics.

In birds, males are the homogametic sex (ZZ) and females the heterogametic sex (ZW). Primary sex determination is thought to depend on a sex chromosome gene dosage mechanism, and the most likely sex determinant is the Z chromosome gene Doublesex and Mab-3-Related Transcription factor 1 (DMRT1). To clarify this issue, we used a CRISPR-Cas9-based monoallelic targeting approach and sterile surrogate hosts to generate birds with targeted mutations in the DMRT1 gene. The resulting chromosomally male (ZZ) chicken with a single functional copy of DMRT1 developed ovaries in place of testes, demonstrating the avian sex-determining mechanism is based on DMRT1 dosage. These ZZ ovaries expressed typical female markers and showed clear evidence of follicular development. However, these ZZ adult birds with an ovary in place of testes were indistinguishable in appearance to wild-type adult males, supporting the concept of cell-autonomous sex identity (CASI) in birds. In experiments where estrogen synthesis was blocked in control ZW embryos, the resulting gonads developed as testes. In contrast, if estrogen synthesis was blocked in ZW embryos that lacked DMRT1, the gonads invariably adopted an ovarian fate. Our analysis shows that DMRT1 is the key sex determination switch in birds and that it is essential for testis development, but that production of estrogen is also a key factor in primary sex determination in chickens, and that this production is linked to DMRT1 expression.

Differential expression of microRNAs in diapause and non-diapause gonads of Aspongopus chinensis Dallas (Hemiptera: Dinidoridae): implications for reproductive control.

Aspongopus chinensis Dallas, 1851 (Hemiptera: Dinidoridae), an edible and medicinal insect, usually found in China and Southeast Asia, offers substantial potential for various applications. The reproductive cycle of this particular insect occurs annually because of reproductive diapause, leading to inadequate utilization of available natural resources. Despite its considerable ecological importance, the precise mechanisms underlying diapause in A. chinensis are not yet well understood. In this study, we conducted an analysis of comparing the microRNA (miRNA) regulation in the diapause and non-diapause gonads of A. chinensis and identified 303 differentially expressed miRNAs, among which, compared with the diapause group, 76 miRNAs were upregulated and 227 miRNAs downregulated. The results, regarding the Enrichment analysis of miRNA-targeted genes, showed their involvement in several essential biological processes, such as lipid anabolism, energy metabolism, and gonadal growth. Interestingly, we observed that the ATP-binding cassette pathway is the only enriched pathway, demonstrating the capability of these targeted miRNAs to regulate the reproductive diapause of A. chinensis through the above essential pathway. The current study provided the role of gonadal miRNA expression in the control of reproductive diapause in A. chinensis, the specific regulatory mechanism behind this event remained unknown and needed more investigation.

Unraveling the spawning and reproductive patterns of tomato hind grouper, Cephalopholis sonnerati (Valenciennes, 1828) from south Kerala waters.

The objective of this study is to provide information on the reproductive biology of tomato hind grouper, Cephalopholis sonnerati (Valenciennes, 1828) for conservation and management purposes. Fish caught by artisanal fishermen from September 2019 to August 2021 were analysed. A total of 280 females, 31 males, and 4 transitional and 178 sex-undetermined fish were analysed. The female to male sex proportion was 9:1, and the fish reached a maximum total body length of 38.5 and 54.5 cm for females and males, respectively. The following microscopic stages were identified: immature, developing, ripe, running ripe/releasing, and spent in both males and females. Several asynchronous development patterns were observed in the studied gonads, including multiple oocyte stages and early and advanced stages of sexual transition. High gonadosomatic index (GSI) for both males and females was recorded in March, May, and November. Running ripe and releasing stages in females were identified in the months from March to June, which indicates the spawning season. The absolute and relative fecundity of the species ranged from 162,723 ± 207,267 and 239 ± 285, respectively. An exponential relationship was found between fecundity and total body length (TL), fecundity and total body weight (TW), and fecundity and gonad weight (GW).

Theoretical Analysis and Expression Profiling of 17ß-Hydroxysteroid Dehydrogenase Genes in Gonadal Development and Steroidogenesis of Leopard Coral Grouper (Plectropomus leopardus).

The differentiation and developmental trajectory of fish gonads, significantly important for fish breeding, culture, and production, has long been a focal point in the fields of fish genetics and developmental biology. However, the mechanism of gonadal differentiation in leopard coral grouper (Plectropomus leopardus) remains unclear. This study investigates the 17ß-Hydroxysteroid Dehydrogenase (Hsd17b) gene family in P. leopardus, with a focus on gene characterization, expression profiling, and functional analysis. The results reveal that the P. leopardus's Hsd17b gene family comprises 11 members, all belonging to the SDR superfamily. The amino acid similarity is only 12.96%, but conserved motifs, such as TGxxxGxG and S-Y-K, are present in these genes. Hsd17b12a and Hsd17b12b are unique homologs in fish, and chromosomal localization has confirmed that they are not derived from different transcripts of the same gene, but rather are two independent genes. The Hsd17b family genes, predominantly expressed in the liver, heart, gills, kidneys, and gonads, are involved in synthesizing or metabolizing sex steroid hormones and neurotransmitters, with their expression patterns during gonadal development categorized into three distinct categories. Notably, Hsd17b4 and Hsd17b12a were highly expressed in the testis and ovary, respectively, suggesting their involvement in the development of reproductive cells in these organs. Fluorescence in situ hybridization (FISH) further indicated specific expression sites for these genes, with Hsd17b4 primarily expressed in germ stem cells and Hsd17b12a in oocytes. This comprehensive study provides foundational insights into the role of the Hsd17b gene family in gonadal development and steroidogenesis in P. leopardus, contributing to the broader understanding of fish reproductive biology and aquaculture breeding.

Role of Mn-LIPA in Sex Hormone Regulation and Gonadal Development in the Oriental River Prawn, Macrobrachium nipponense.

This study investigates the role of lysosomal acid lipase (LIPA) in sex hormone regulation and gonadal development in Macrobrachium nipponense. The full-length Mn-LIPA cDNA was cloned, and its expression patterns were analyzed using quantitative real-time PCR (qPCR) in various tissues and developmental stages. Higher expression levels were observed in the hepatopancreas, cerebral ganglion, and testes, indicating the potential involvement of Mn-LIPA in sex differentiation and gonadal development. In situ hybridization experiments revealed strong Mn-LIPA signaling in the spermatheca and hepatopancreas, suggesting their potential role in steroid synthesis (such as cholesterol, fatty acids, cholesteryl ester, and triglycerides) and sperm maturation. Increased expression levels of male-specific genes, such as insulin-like androgenic gland hormone (IAG), sperm gelatinase (SG), and mab-3-related transcription factor (Dmrt11E), were observed after dsMn-LIPA (double-stranded LIPA) injection, and significant inhibition of sperm development and maturation was observed histologically. Additionally, the relationship between Mn-LIPA and sex-related genes (IAG, SG, and Dmrt11E) and hormones (17ß-estradiol and 17α-methyltestosterone) was explored by administering sex hormones to male prawns, indicating that Mn-LIPA does not directly control the production of sex hormones but rather utilizes the property of hydrolyzing triglycerides and cholesterol to provide energy while influencing the synthesis and secretion of self-sex hormones. These findings provide valuable insights into the function of Mn-LIPA in M. nipponense and its potential implications for understanding sex differentiation and gonadal development in crustaceans. It provides an important theoretical basis for the realization of a monosex culture of M. nipponense.

Gonadal Development and Differentiation of Hybrid F1 Line of Ctenopharyngodon idella (♀) × Squaliobarbus curriculus (♂).

The hybrid F1 offspring of Ctenopharyngodon idella (♂) and Squaliobarbus curriculus (♀) exhibit heterosis in disease resistance and also show abnormal sex differentiation. To understand the mechanism behind gonadal differentiation in the hybrid F1, we analyzed the transcriptomes of C. idella, S. curriculus, and the hybrid F1; screened for genes related to gonad development in these samples; and measured their expression levels. Our results revealed that compared to either C. idella or S. curriculus, the gene expressions in most sub-pathways of the SNARE interactions in the vesicular transport pathway in the hypothalamus, pituitary, and gonadal tissues of their hybrid F1 offspring were significantly up-regulated. Furthermore, insufficient transcription of genes involved in oocyte meiosis may be the main reason for the insufficient reproductive ability of the hybrid F1 offspring. Through transcriptome screening, we identified key molecules involved in gonad development, including HSD3B7, HSD17B1, HSD17B3, HSD20B2, CYP17A2, CYP1B1, CYP2AA12, UGT2A1, UGT1A1, and FSHR, which showed significant differences in expression levels in the hypothalamus, pituitary, and gonads of these fish. Notably, the expression levels of UGT1A1 in the gonads of the hybrid F1 were significantly higher than those in C. idella and S. curriculus. These results provide a scientific basis for further research on the gonadal differentiation mechanism of hybrid F1 offspring.

Genome-Wide Identification and Expression Analysis of the BTB Gene Superfamily Provides Insight into Sex Determination and Early Gonadal Development of Alligator sinensis.

The BTB gene superfamily is widely distributed among higher eukaryotes and plays a significant role in numerous biological processes. However, there is limited knowledge about the structure and function of BTB genes in the critically endangered species Alligator sinensis, which is endemic to China. A total of 170 BTB genes were identified from the A. sinensis genome, classified into 13 families, and unevenly distributed across 16 chromosomes. Analysis of gene duplication events yielded eight pairs of tandem duplication genes and six pairs of segmental duplication genes. Phylogenetics shows that the AsBTB genes are evolutionarily conserved. The cis-regulatory elements in the AsBTB family promoter region reveal their involvement in multiple biological processes. Protein interaction network analysis indicates that the protein interactions of the AsBTB genes are centered around CLU-3, mainly participating in the regulation of biological processes through the ubiquitination pathway. The expression profile and protein interaction network analysis of AsBTB genes during sex differentiation and early gonadal development indicate that AsBTB genes are widely expressed in this process and involves numerous genes and pathways for regulation. This study provides a basis for further investigation of the role of the BTB gene in sex differentiation and gonadal development in A. sinensis.

Effects of exogenous steroid hormones on growth, body color, and gonadal development in the Opsariichthys bidens.

To investigate the effects of exogenous steroid hormones on growth, body color, and gonadal development in the Opsariichthys bidens (O. bidens), synthetic methyltestosterone (MT) and 17ß-estradiol (E2) were used for 28 days' treatment of 4-month-old O. bidens before the breeding season. Our results suggested that MT had a significant growth-promoting effect (P < 0.05), whereas E2 played an inhibitory role. On the body surface, the females in the MT group showed gray stripes, and the fish in other groups showed no obvious stripes. The males with MT treatment displayed brighter blue-green stripes compared to the CK and E2 groups. The histological analysis showed that the MT significantly promoted testes development in males, blocked oocyte development, and caused massive apoptosis in females, whereas the E2 group promoted ovarian development and inhibited testes development. Based on qRT-PCR analysis, in females, the expression of igf-1, dmrt1, and cyp19a1a genes revealed that E2 treatment resulted in down-regulation of igf-1 expression and up-regulation of cyp19a1a expression. In males, igf-1 and dmrt1 were significantly up-regulated after MT treatment, and E2 treatment led to down-regulation of igf-1. Therefore, this study demonstrates that MT and E2 play an important role in reversing the morphological sex characteristics of females and males.

Genetic evidence for Amh modulation of gonadotropin actions to control gonadal homeostasis and gametogenesis in zebrafish and its noncanonical signaling through Bmpr2a receptor.

Anti-Müllerian hormone (Amh) plays an important role in gonadal function. Amh deficiency causes severe gonadal dysgenesis and dysfunction in zebrafish, with gonadal hypertrophy in both sexes. However, its mechanism of action remains unknown. Intriguingly, the Amh cognate type II receptor (Amhr2) is missing in the zebrafish genome, in sharp contrast to other species. Using a series of zebrafish mutants (amh, fshb, fshr and lhcgr), we provided unequivocal evidence for actions of Amh, via modulation of gonadotropin signaling, on both germ cell proliferation and differentiation. The gonadal hypertrophy in amh mutants was abolished in the absence of Fshr in females or Fshr/Lhcgr in males. Furthermore, we demonstrated that knockout of bmpr2a, but not bmpr2b, phenocopied all phenotypes of the amh mutant in both sexes, including gonadal hypertrophy, hyperproliferation of germ cells, retarded gametogenesis and reduced fshb expression. In summary, the present study provided comprehensive genetic evidence for an intimate interaction of gonadotropin and Amh pathways in gonadal homeostasis and gametogenesis and for Bmpr2a as the possible missing link for Amh signaling in zebrafish.

Molecular cloning and expression patterns of a sex-biased transcriptional factor Foxl2 in the giant freshwater prawn (Macrobrachium rosenbergii).

BACKGROUD: Macrobrachium rosenbergii is an economically important species that is widely cultivated in some Asian nations. Foxl2 is a transcriptional regulator of ovarian differentiation and development. The aim of this study was to study the bioinformatics features and expression patterns of M. rosenbergii Foxl2 (MrFoxl2). METHODS: In this study, all experimental animals were mature M. rosenbergii (9-12 cm) individuals. The foxl2 gene was identified and characterized in the genome of M. rosenbergii using molecular cloning, bioinformatic analysis, in situ hybridization, and quantitative analysis. RESULTS: The identified cDNA encoded a putative 489-amino-acid MrFoxl2 protein. Bioinformatics analysis revealed a low identity of MrFoxl2 to other crustacean orthologues. The closest phylogenetic relationship was to Foxl2 of Eriocheir sinensis. The result of in situ hybridization demonstrated that transcripts of MrFoxl2 in M. rosenbergii were identified in spermatocytes, oocytes, and secretory epithelial cells of the vas deferens. The result of q-PCR suggested that a high expression of MrFoxl2 was identified in the testis, vas deferens, and ovaries. During ovarian development, MrFoxl2 expression was the highest in stage I. CONCLUSION: Our findings suggest that MrFoxl2 may play a role in gonadal development in both female and male M. rosenbergii.

Genome-wide identification, phylogeny, expression and eyestalk neuroendocrine regulation of vitellogenin gene family in the freshwater giant prawn Macrobrachium rosenbergii.

Vitellogenin (Vg) is the precursor of vitellin, which is an important female-specific protein stored in oocytes as the major nutrient and energy sources for embryogenesis in oviparous animals. In this study, we performed comprehensive genome-wide analysis of Vg gene family in the prawn Macrobrachium rosenbergii, and eight Vg genes designated as MrVg1a, MrVg1b and MrVg2-7 were identified. MrVg1a clusters with the previously described MrVg1b near the end of chromosome 46 and MrVg2 is on the chromosome 42 while MrVg3-7 cluster on the chromosome 23. All the putative MrVg proteins are characterized by the presence of three conserved functional domains: LPD-N, DUF1943 and vWD. Phylogenetic analysis revealed that MrVg1a shares 93% identity with MrVg1b and groups together into a branch while MrVg2-7 group into another branch, suggesting that MrVg1a, 1b and MrVg2-7 might diversify from a common ancestral gene. All the corresponding MrVg transcripts especially for MrVg1 exhibit high expression in the female hepatopancreas at late vitellogensis stage but extremely low in the ovaries except MrVg5, indicating that hepatopancreas is the major site of MrVgs synthesis. In the male, interestingly, MrVg5 and MrVg6 are also highly expressed in the testis, suggesting their potential involvement in testicular development. Bilateral ablation of eyestalk significantly upregulate all the MrVgs mRNA in the female hepatopancreas and the MrVg1 in ovary, but have no effect on the expression of MrVg2-7 in the ovary, demonstrating that eyestalk hormones could promote the ovarian development mostly by inducing the synthesis of MrVgs in the hepatopancreas but rarely in the ovary. Our results provide new insights into the prawn MrVgs family and improve our understanding of the potential role for each member of the family in the gonadal development of M. rosenbergii.

Prevalence and effects of a parasitic trematode on the blue mussel, Mytilus edulis, in the Boston Harbor.

The blue mussel, Mytilus edulis, is a keystone species in the North Atlantic that plays critical roles in nutrient cycling, water filtration, and habitat creation. Blue mussel populations have declined significantly throughout the North Atlantic due to various factors, including habitat loss, pollution, increasing water temperature, and parasites. One parasite is Proctoeces maculatus, a digenetic trematode, which uses M. edulis as an intermediate host. This parasite causes reduced growth, castration, and death in mussels. The range of P. maculatus has expanded northward from Cape Cod, MA to Maine which may be associated with rising temperatures in the Gulf of Maine. To evaluate the negative impacts of P. maculatus on mussels, we analyzed its infections in M. edulis throughout the Boston Harbor, MA. P. maculatus was present in every population and time point analyzed, with approximately 50% of mussels in the harbor infected. The parasite reduced gonadal development in infected mussels, which could lead to decreased fecundity. Severe P. maculatus infections induced a stress response, indicated by increased HSP70 expression. We developed a non-destructive hemolymph-based assay to determine if mussels are infected with P. maculatus, thus speeding up the evaluation process and eliminating the need to sacrifice individuals. With P. maculatus' continued expansion northward, more mussel populations will be under threat from this parasite.

Transcriptomic response to GnRH down regulation by RNA interference in clam Ruditapes philippinarum, suggest possible role in reproductive function.

Gonadotropin-releasing hormone (GnRH) plays a key role in the control of the reproductive axis in vertebrates, however, little is known about its function in reproductive endocrine regulation in molluscs. In the present study, RNA-seq was used to construct transcriptomes of Ruditapes philippinarum testis and ovaries of control and GnRH suppressed individuals using RNA interference. GnRH suppression caused 112 and 169 enriched KEGG pathways in testis and ovary, with 92 pathways in common in both comparisons. The most enriched KEGG pathways occurred in the "Oxidative phosphorylation", "Dorso-ventral axis formation", "Thyroid hormone synthesis" and "Oxytocin signaling pathway" etc. A total of 1838 genes in testis and 358 genes in ovaries were detected differentially expressed in GnRH suppressed clams. Among the differentially expressed genes, a suit of genes related to regulation of steroid hormones synthesis and gonadal development, were found in both ovary and testis with RNAi of GnRH. These results suggest that GnRH may play an important role in reproductive function in bivalves. This study provides a preliminary basis for studying the function and regulatory mechanism of GnRH in bivalves.

Long-day stimulation increases thyroid-stimulating hormone expression and affects gonadal development in chub mackerel.

For seasonal breeders, photoperiodic changes are important signals that mark the start of the breeding season. Thyroid-stimulating hormone (TSH) is a glycoprotein hormone that not only promotes the secretion of thyroid hormone but also plays a key role in regulating seasonal reproduction in birds and mammals. However, whether TSH activation has been implicated as a seasonal indicator in fish breeding has not been fully investigated. In this study, we isolated tshb as a starting point to elucidate the effect of photoperiodic changes on the activation of the reproductive axis of chub mackerel. The isolated tshb was classified as tshba, which is widely conserved in vertebrates. The quantitative PCR results showed that tshb was strongly expressed in the pituitary. When female and male chub mackerel with immature gonads were reared for six weeks under different photoperiodic conditions, the gonads developed substantially in the long-day (LD) reared fish compared to those in the short-day reared fish. Real-time PCR results showed that the expression level of tshb in the pituitary gland was significantly elevated in the LD group. Although there was no difference in the gonadotropin-releasing hormone 1 gene expression level in the preoptic area of the brain, follicle-stimulating hormone and luteinizing hormone gene expression levels in the pituitary were also significantly elevated in the LD group. In conclusion, TSH is a potential mediator of seasonal information in the reproductive endocrine axis and may induce gonadal development during the breeding season of chub mackerel.

Steroidogenic factor 1 (NR5A1) induces multiple transcriptional changes during differentiation of human gonadal-like cells.

Nuclear receptor subfamily 5 group A member 1 (NR5A1) encodes steroidogenic factor 1 (SF1), a key regulatory factor that determines gonadal development and coordinates endocrine functions. Here, we have established a stem cell-based model of human gonadal development and applied it to evaluate the effects of NR5A1 during the transition from bipotential gonad to testicular cells. We combined directed differentiation of human induced pluripotent stem cells (46,XY) with activation of endogenous NR5A1 expression by conditionally-inducible CRISPR activation. The resulting male gonadal-like cells expressed several Sertoli cell transcripts, secreted anti-Müllerian hormone and responded to follicle-stimulating hormone by producing sex steroid intermediates. These characteristics were not induced without NR5A1 activation. A total of 2691 differentially expressed genetic elements, including both coding and non-coding RNAs, were detected immediately following activation of NR5A1 expression. Of those, we identified novel gonad-related putative NR5A1 targets, such as SCARA5, which we validated also by immunocytochemistry. In addition, NR5A1 activation was associated with dynamic expression of multiple gonad- and infertility-related differentially expressed genes. In conclusion, by combining targeted differentiation and endogenous activation of NR5A1 we have for the first time, been able to examine in detail the effects of NR5A1 in early human gonadal cells. The model and results obtained provide a useful resource for future investigations exploring the causative reasons for gonadal dysgenesis and infertility in humans.

Improved biomarker discovery through a plot twist in transcriptomic data analysis.

BACKGROUND: Transcriptomic analysis is crucial for understanding the functional elements of the genome, with the classic method consisting of screening transcriptomics datasets for differentially expressed genes (DEGs). Additionally, since 2005, weighted gene co-expression network analysis (WGCNA) has emerged as a powerful method to explore relationships between genes. However, an approach combining both methods, i.e., filtering the transcriptome dataset by DEGs or other criteria, followed by WGCNA (DEGs + WGCNA), has become common. This is of concern because such approach can affect the resulting underlying architecture of the network under analysis and lead to wrong conclusions. Here, we explore a plot twist to transcriptome data analysis: applying WGCNA to exploit entire datasets without affecting the topology of the network, followed with the strength and relative simplicity of DEG analysis (WGCNA + DEGs). We tested WGCNA + DEGs against DEGs + WGCNA to publicly available transcriptomics data in one of the most transcriptomically complex tissues and delicate processes: vertebrate gonads undergoing sex differentiation. We further validate the general applicability of our approach through analysis of datasets from three distinct model systems: European sea bass, mouse, and human. RESULTS: In all cases, WGCNA + DEGs clearly outperformed DEGs + WGCNA. First, the network model fit and node connectivity measures and other network statistics improved. The gene lists filtered by each method were different, the number of modules associated with the trait of interest and key genes retained increased, and GO terms of biological processes provided a more nuanced representation of the biological question under consideration. Lastly, WGCNA + DEGs facilitated biomarker discovery. CONCLUSIONS: We propose that building a co-expression network from an entire dataset, and only thereafter filtering by DEGs, should be the method to use in transcriptomic studies, regardless of biological system, species, or question being considered.

Same-sex Pairs Retain Their Reproductive Capacity as a Potential Opportunity for Individual Reproductive Success in Termites.

In eusocial termites, successful pairing is an essential element of dispersal and distribution after the departure of alates from natal colonies. Two situations could arise during the pairing process: mixed-sex pairs and same-sex pairs. However, most previous studies focused on mixed-sex pairs, overlooking groups formed by same-sex pairings, especially potential fecundity (the total number of oocytes or ovarioles), oogenesis and the development stage of oocytes of females in female-female pairs, and spermatogenesis and testis development of males in male-male pairs. In this study, through experimentation, we investigated the reproductive ability of virgin dealates based on various pairing types as mentioned above. We found that the life spans of virgin dealates can cover 1 yr or even more when they establish a nest with a partner, which is more than 10-fold longer than the life span of individuals establishing a colony alone. After 1 yr of pairing, the potential fecundity of virgin same sex dealates did not degenerate significantly compared with newly emerged dealates, including the number of ovarioles, size of testis, oogenesis, and the development stage of the oocytes. Moreover, when individuals of same-sex pairings experimentally changed into mixed-sex pairs after 1 yr, the eggs produced in the colony hatched into larvae. These findings suggest that dealates which through same-sex pairs retain fecundity after 1 yr have more reproductive potential than dealates that failed to pair with heterosexuals, shedding light on the ecological significance of homosexual behaviors in terms of the successful extension and fecundity of eusocial termites.