Structure and function of vasa gene in gonadal gametogenesis of Pacific abalone.

Pacific abalone (Haliotis discus hannai) is a marine gastropod mollusc with significant economic importance in both global fisheries and aquaculture. However, studies exploring the gonadal development and regulatory mechanisms of Haliotis discus hannai are limited. This study aimed to explore whether the vasa gene acted as a molecular marker for germ cells. Initially, the vasa gene was successfully cloned using the cDNA-end rapid amplification technique. The cloned gene had a 2478-bp-long open reading frame and encoded 825 amino acids. Then, a recombinant expression vector was constructed based on the Vasa protein, and an 87-kDa recombinant protein was prepared. Subsequently, a polyclonal antibody was prepared using the purified recombinant protein. The enzyme-linked immunosorbent assay (ELISA) confirmed the titer of the antibody to be ≥512 K. The immunohistochemical analysis revealed that Vasa was widely expressed in oogonia, Stage I oocytes, spermatogonia, and primary spermatocytes. The specific expression of Vasa in the hermaphroditic gonads of abalone was assessed using western blotting to investigate the effects of different photoperiods (12 L:12D, 24 L:0D, 18 L:6D, and 6 L:18D) on the gonadal development of abalone (P < 0.05), with higher expression levels observed in the ovarian proliferative and spermary maturing stages compared with other developmental stages (P < 0.05). Additionally, Vasa exhibited the highest expression in the spermary and ovary under a photoperiod of 18 L:6D (P < 0.05). These data demonstrated the key role of Vasa in developing germ cells in abalone. They shed light upon the molecular mechanism through which the photoperiod influenced Vasa expression and regulated gonadal development in abalone. The findings might provide theoretical references for analyzing the differentiation pattern of abalone germ cells and the genetic improvement and conservation of germplasm resources.

Tracking gonadal development in fish: An in vivo MRI study on polar cod, Boreogadus saida (Lepechin, 1774).

Magnetic resonance imaging (MRI) was applied to determine the sex of polar cod (Boreogadus saida Lepechin, 1774) (Actinopterygii: Gadidae) and to follow the gonadal development in individual animals over time. Individual unanaesthetised fish were transferred to a measurement chamber inside a preclinical 9.4 T MRI scanner and continuously perfused with aerated seawater. A screening procedure at an average of 3.5 h, consisting of a set of MRI scans of different orientations, was repeated every 4 weeks on the same set of reproducing B. saida (n = 10) with a body length of about 20 cm. Adapted multi-slice flow-compensated fast low-angle shot (FcFLASH) and rapid acquisition with relaxation enhancement (RARE) protocols with an in-plane resolution of 313 µm and an acquisition time of 2.5 min were used to visualise the morphology of various organs, including the gonads within the field of view (FOV). The MR images provided high resolution, enabling specific sex determination, calculation of gonad volumes, and determination of oocyte sizes. Gonad maturation was followed over 4 months from November 2021 until shortly before spawning in February 2022. The gonad volume increased by 2.3-25.5% for males and by 11.5-760.7% for females during the observation period. From October to February, the oocyte diameter increased from 427 µm (n = 1) to 1346 ± 27 µm (n = 4). Interestingly, individual oocytes showed changes in MR contrast over time that can be attributed to the morphological development of the oocytes. The results fit well with previous literature data from classical invasive studies. The presented approach has great potential for various ecophysiological applications such as monitoring natural or delayed development of internal organs or sex determination under different environmental conditions.

Alterations in neurotransmitters, steroid hormones, vitellogenin, and antioxidant system induced by di-n-butyl phthalate and di-isopentyl phthalate on catfish Rhamdia quelen.

Phthalates, such as di-n-butyl phthalate (DBP) and di-isopentyl phthalate (DiPeP), are pollutants with a high potential for endocrine disruption. This study aimed to evaluate parameters of endocrine disruption in specimens of the Neotropical fish Rhamdia quelen exposed to DBP and DiPeP through their food. After 30 days of exposure, the fish were anesthetized and then euthanized, and blood, hypothalamus, liver, and gonads were collected. DBP caused statistically significant alterations in the serotoninergic system of males (5 and 25 ng/g) and females (5 ng/g) of R. quelen and it increased testosterone levels in females (25 ng/g). DiPeP significantly altered the dopaminergic system in females, reduced plasma estradiol levels (125 ng/g) and hepatic vitellogenin expression (25 ng/g), and changed the antioxidant system in gonads (125 ng/g). The results suggest that DBP and DiPeP may have different response patterns in females, with the former being androgenic and the latter being anti-estrogenic. These findings provide additional evidence regarding the molecular events involving DBP and DiPeP in the endocrine disruption potential in juvenile specimens of Rhamdia quelen.

Genome-wide analysis of the cytochrome P450 gene family in Pacific oyster Crassostrea gigas and their expression profiles during gonad development.

The cytochrome P450 (CYP) gene superfamily plays a significant role in various physiological processes, producing different compounds such as hormones, fatty acids, and biomolecules. However, little information is known their roles during gonad development in Pacific oyster (Crassostrea gigas). In this study, total of 116 CgCYP (Crassostrea gigas cytochrome P450) genes were identified and their expression pattern was analyzed for the first time. The relative molecular weights of these CgCYP genes ranged from 63.52 to 113.41 kDa, and the length of encoded amino acids ranged from 103 to 993. And total 26 cis-acting elements of these CgCYP genes were identified. GO and KEGG enrichment analysis showed some CgCYP genes are essential for the metabolism of male and female sex hormones. Additionally, expression anslysis showed 69 CgCYP genes were over-expressed in early gonad development and triploid infertile individuals. More importantly, expression levels of CgCYP1, CgCYP15, CgCYP34, CgCYP46, CgCYP69, CgCYP87, CgCYP88, and CgCYP103, were found to be significantly higher in female gonad, suggesting their important roles in female gonad development. The results of this study will provide a better understanding of the CgCYP genes in the gonad development of Pacific oyster.

Investigation of Sexes and Fertility Potential of Female Russian Sturgeon (Acipenser gueldenstaedtii) and Male American Paddlefish (Polyodon spathula) Hybrids.

In the present study, 10 allotriploid (3nALT) and 10 allopentaploid (5nALP) six-month-old hybrid fish and two 3nALT and four 5nALP 40-month-old hybrid fish, which resulted by crossing female Russian sturgeon Acipenser gueldenstaedtii (Brandt and Ratzeberg, 1833) and male American paddlefish Polyodon spathula (Walbaum, 1792), were investigated. It was revealed that six-month-old 3nALT and 5nALP hybrids initially had "undifferentiated" gonads, while in the 40-month-old hybrids, only testes were observed in one case of 3nALT and one case of 5nALP hybrids. The testis of 3nALT hybrids was partially developed with spermatogonia, while the testis of one 5nALP hybrid was in the second developmental stage with low spermatogonia density. We could not determine gonad differentiation in any of the cases when the hybrid individuals had the W sex chromosome. We concluded that the gonad differentiation of these interfamilial hybrids follows a similar pattern to interspecific hybrids of different ploidy parent species of the family Acipenseridae, which is consistent with the classical Haldane's rule. However, it cannot be excluded that the testis of this/these hybrid(s) may produce fertile sperm after sexual maturity, depending on additional genetic, hormonal and environmental factors, and further research is required for its evaluation.

Expression of Tshb and Tshr in the ricefield eel Monopterus albus: Potential paracrine/autocrine roles in gonads.

Thyroid stimulating hormone (TSH), a glycoprotein synthesized and secreted from thyrotrophs of the pituitary gland, is composed of a glycoprotein hormone common alpha subunit (CGA) and a specific beta subunit (TSHB). The major biological function of TSH is to stimulate thyroidal follicles to synthesize and secrete thyroid hormones through activating its cognate receptor, the thyroid stimulating hormone receptor (TSHR). In the present study, polyclonal antisera against ricefield eel Tshb and Tshr were generated respectively, and the expression of Tshb and Tshr was examined at mRNA and protein levels. RT-PCR analysis showed that tshb mRNA was expressed mainly in the pituitary as well as in some extrapituitary tissues including the ovary and testis. Tshr mRNA was also expressed in a tissue-specific manner, with transcripts detected in tissues including the kidney, ovary, and testis. The immunoreactive Tshb signals in the pituitary were shown to be localized to the inner areas of adenohypophysis which are close to the neurohypophysis of adult ricefield eels. Tshb-immunoreatvie cells in the pituitary of ricefield eel larvae were firstly observed at hatching. The expression of immunoreactive Tshb and Cga was also detected in ricefield eel ovary and testis together with Tshr. In the ovary, immunoreactive Tshb, Cga, and Tshr were observed in oocytes and granulosa cells. In the testis, immunoreactive Tshb was mainly observed in Sertoli cells while immunoreactive Cga and Tshr were detected in germ cells as well as somatic cells. Results of the present study suggest that Tsh may be synthesized both in the ovary and testis locally, which may play paracrine and/or autocrine roles in gonadal development in ricefield eels.

Effects of whole life-cycle exposure to carbaryl on reproduction of female marine medaka (Oryzias melastigma) and their offspring.

Carbaryl is widely used as a highly effective insecticide which harms the marine environment. This study aimed to assess the reproductive toxicity of chronic carbaryl exposure on female marine medaka and their female offspring. After a 180-day exposure from embryonic period to adulthood, females exhibited reduced attraction to males, decreased ovulation, increased gonadosomatic index and a higher proportion of mature and atretic follicles. These reproductive toxic effects of carbaryl may stem from changes in hormone levels and transcription levels of key genes along the HPG axis. Furthermore, maternal carbaryl exposure had detrimental effects on the offspring. F1 females showed the reproductive disorders similar to those observed in F0 females. The significant changes in the transcription levels of DNA methyltransferase and demethylase genes in the F0 and F1 generations of ovaries indicate changes in their DNA methylation levels. The changes in DNA methylation levels in F1 female marine medaka may lead to changes in the expression of certain reproductive key genes, such as an increase in the transcription level of cyp19a, which may be the reason for F1 reproductive toxicity. These findings indicate that maternal exposure may induce severe generational toxicity through alterations in DNA methylation levels. This study assesses the negative impacts of whole life-cycle carbaryl exposure on the reproductive and developmental processes of female marine medaka and its female offspring, while offering data to support the evaluation of the ecological risk posed by carbaryl in marine ecosystems.

Human Gonads Do Not Contribute to the Circulating Pool of 11-Oxygenated Androgens.

CONTEXT: Androstenedione (A4) and testosterone (T) are produced by both the adrenal glands and the gonads. The adrenal enzyme 11ß-hydroxylase (CYP11B1) executes the final step in cortisol synthesis; CYP11B1 also uses A4 and T as substrates, generating 11-hydroxyandrostenedione and 11-hydroxytestosterone, respectively. It has been suggested that CYP11B1 is expressed in the gonads, yet the circulating levels of all 11-oxygenated androgens (11-oxyandrogens) are similar in males and females of reproductive ages, despite enormous differences in T. OBJECTIVE: To assess the gonadal contribution to the circulating pool of 11-oxyandrogens. METHODS: We used liquid chromatography-tandem mass spectrometry to measure 13 steroids, including traditional and 11-oxyandrogens in: (I) paired gonadal and peripheral vein blood samples obtained during gonadal venograms from 11 patients (7 women), median age 37 (range 31-51 years); and (II) 17 women, median age 57 (range 41-81 years) before and after bilateral salpingo-oophorectomy (BSO). We also compared CYP11B1, 17α-hydroxylase/17,20-lyase (CYP17A1), and 3ß-hydroxysteroid dehydrogenase type 2 (HSD3B2) mRNA expression in adrenal, ovarian, and testicular tissue. RESULTS: A4, T, estradiol, estrone, progesterone, 17α- and 16α-hydroxyprogesterone were all higher in gonadal veins vs. periphery (p < 0.05 for all), while four 11-oxyandrogens were similar between matched gonadal and peripheral vein samples. Equally, in women who underwent BSO, A4 (median [interquartile range]: 59.7 [47.7-67.6] ng/dL vs. 32.7 [27.4-47.8] ng/dL, p < 0.001) and T (24.1 [16.4-32.3] vs.15.5 [13.7-19.0] ng/dL, p < 0.001) declined, while 11-oxyandrogens remained stable. Gonadal tissue displayed negligible CYP11B1 mRNA. CONCLUSION: Despite producing substantial amounts of A4 and T, human gonads are not relevant sources of 11-oxyandrogens.

Exploring the co-exposure effects of environmentally relevant microplastics and an estrogenic mixture on the metabolome of the Sydney rock oyster.

In aquatic environments the concurrent exposure of molluscs to microplastics (MPs) and estrogens is common, as these pollutants are frequently released by wastewater treatment plants into estuaries. Therefore, this study aimed to evaluate the independent and co-exposure impacts of polyethylene microplastics (PE-MPs) and estrogenic endocrine-disrupting chemicals (EEDCs) at environmentally relevant concentrations on polar metabolites and morphological parameters of the Sydney rock oyster. A seven-day acute exposure revealed no discernible differences in morphology; however, significant variations in polar metabolites were observed across oyster tissues. The altered metabolites were mostly amino acids, carbohydrates and intermediates of the Kreb's cycle. The perturbation of metabolites were tissue and sex-specific. All treatments generally showed an increase of metabolites relative to controls - a possible stimulatory and/or a potential hormetic response. The presence of MPs impeded the exposure of adsorbed and free EEDCs potentially due to the selective feeding behaviour of oysters to microplastics, favouring algae over similar-sized PE-MPs, and the formation of an eco/bio-corona involving faeces, pseudo-faeces, natural organic matter, and algae.

Status of single-cell RNA sequencing for reproductive toxicology in zebrafish and the transcriptomic trade-off.

The utilization of transcriptomic studies identifying profiles of gene expression, especially in toxicogenomics, has catapulted next-generation sequencing to the forefront of reproductive toxicology. An innovative yet underutilized RNA sequencing technique emerging into this field is single-cell RNA sequencing (scRNA-seq), which provides sequencing at the individual cellular level of gonad tissue. ScRNA-seq provides a novel and unique perspective for identifying distinct cellular profiles, including identification of rare cell subtypes. The specificity of scRNA-seq is a powerful tool for reproductive toxicity research, especially for translational animal models including zebrafish. Studies to date not only have focused on 'tissue atlassing' or characterizing what cell types make up different tissues but have also begun to include toxicant exposure as a factor that this review aims to explore. Future scRNA-seq studies will contribute to understanding exposure-induced outcomes; however, the trade-offs with traditional methods need to be considered.

Two C. elegans DM domain proteins, DMD-3 and MAB-3, function in late stages of male somatic gonad development.

To bring about sexual dimorphism in form, information from the sex determination pathway must trigger sex-specific modifications in developmental programs. DM-domain encoding genes have been found to be involved in sex determination in a multitude of animals, often at the level of male somatic gonad formation. Here we report our findings that the DM-domain transcription factors MAB-3 and DMD-3 function together in multiple steps during the late stages of C. elegans male somatic gonad development. Both mab-3 and dmd-3 are expressed in the linker cell and hindgut of L4 males and dmd-3 is also expressed in presumptive vas deferens cells. Furthermore, dmd-3, but not mab-3, expression in the linker cell is downstream of nhr-67, a nuclear hormone receptor that was previously shown to control late stages of linker cell migration. In mab-3; dmd-3 double mutant males, the last stage of linker cell migration is partially defective, resulting in aberrant linker cell shapes and often a failure of the linker cell to complete its migration to the hindgut. When mab-3; dmd-3 double mutant linker cells do complete their migration, they fail to be engulfed by the hindgut, indicating that dmd-3 and mab-3 activity are essential for this process. Furthermore, linker cell death and clearance are delayed in mab-3; dmd-3 double mutants, resulting in the linker cell persisting into adulthood. Finally, DMD-3 and MAB-3 function to activate expression of the bZIP transcription factor encoding gene zip-5 and downregulate the expression of the zinc metalloprotease ZMP-1 in the linker cell. Taken together, these results demonstrate a requirement for DM-domain transcription factors in controlling C. elegans male gonad formation, supporting the notion that the earliest DM-domain genes were involved in male somatic gonad development in the last common ancestor of the bilaterians.

Asterias Rolleston starfish gonad lipids: A novel source of Omega-3 fatty acids - assessment of major components and their antioxidant activities.

The major lipids and antioxidant activities of Asterias rolleston gonad lipids were evaluated systematically. Major lipids of A. Rolleston gonad lipids were triacylglycerols (TAGs) and phospholipids (PLs). Total lipids were composed of 15.62% of polyunsaturated fatty acids (PUFAs), and 40.81% of monounsaturated fatty acids (MUFAs). The most abundant PUFA were C20:5n-3 (EPA) (6.28%) and C22:6n-3 (DHA) (5.80%). Predominantly composed of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), polar lipids were rich in PUFAs and could contain up to 34.59% EPA and DHA, and PE and PI (phosphatidylinositol) were also found to be the main carriers of EPA and ARA (arachidonic acid) in polar lipids. The MUFA and PUFA of Sn-2 in TAG are 39.72% and 30.37%, respectively. A total of 64 TAG species were identified, with Eo-P-M, Eo-Eo-M, and M-M-Eo being the main TAGs components. Moreover, A. rollestoni gonad lipids exhibited potent radical scavenging activities and reducing power in a dose-dependent manner.

Genome-wide identification and expressional profile of the Dmrt gene family in the swimming crab (Portunus trituberculatus).

The swimming crab, Portunus trituberculatus is one of crucial aquaculture crabs with significant differences in growth and economic performance between male and female swimming crabs. Consequently, the culture of female populations presents higher economic value. The doublesex and mab-3 related transcription factor (Dmrt) gene family are known to play crucial role in gonad differentiation and development. However, there is limited information about this gene family in Portunus trituberculatus. In this study, we identified seven members of the Dmrt gene family in P. trituberculatus based on the published transcriptome and genome data and designated as Ptdmrt-1, Ptdoublesex (Ptdsx), Ptidmrt-1, Ptdmrt-11E, Ptidmrt-2, Ptdmrt-99B, and Ptdmrt-3 based on the homology analysis results, respectively. These Ptdmrt genes distributed across 6 chromosomes and were predicted to encode 283 aa, 288 aa, 529 aa, 436 aa, 523 aa, 224 aa, and 435 aa protein precursors, respectively. The expression patterns of these dmrt genes were characterized by qRT-PCR and gonad transcriptome data. The results showed that five members (Ptdmrt-99B, Ptdsx, Ptdmrt-1, Ptdmrt-3, and Ptdmrt-11E) were differentially expressed between the testis and ovary. In addition, their expression patterns from zoea 2 to juvenile 1 were characterized by published transcriptome data and the results showed that they were lowly expressed and did not exhibit notable difference except for Ptdsx during early development. Overall, majority of Ptdmrt genes were involved in gonad differentiation in the swimming crab. Current findings provide a solid foundation for further exploration of the roles of these genes in gonad development and differentiation in P. trituberculatus.

The single-cell chromatin landscape in gonadal cell lineage specification.

Gonad development includes sex determination and divergent maturation of the testes and ovaries. Recent advances in measuring gene expression in single cells are providing new insights into this complex process. However, the underlying epigenetic regulatory mechanisms remain unclear. Here, we profiled chromatin accessibility in mouse gonadal cells of both sexes from embryonic day 11.5 to 14.5 using single-cell assay for transposase accessible chromatin by sequencing (scATAC-seq). Our results showed that individual cell types can be inferred by the chromatin landscape, and that cells can be temporally ordered along developmental trajectories. Integrative analysis of transcriptomic and chromatin-accessibility maps identified multiple putative regulatory elements proximal to key gonadal genes Nr5a1, Sox9 and Wt1. We also uncover cell type-specific regulatory factors underlying cell type specification. Overall, our results provide a better understanding of the epigenetic landscape associated with the progressive restriction of cell fates in the gonad.

Effects of chronic exposure to a high fat diet, nutritive or non-nutritive sweeteners on hypothalamic-pituitary-adrenal (HPA) and -gonadal (HPG) axes of male Sprague-Dawley rats.

PURPOSE: Diet-related factors are of great significance in the regulation of hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonad (HPG) axes. In this study, we aimed to investigate the effects of chronic exposure to a high fat diet (HFD), fructose or sucralose on the endocrine functions. METHODS: Male, Sprague-Dawley rats received a normal chow diet, HFD, 10% fructose or 0.02% sucralose for 10 weeks. Behavioral changes were assessed by open field (OFT) and elevated plus-maze (EPM) tests at week 8. H&E staining was used to observe pathological changes in adrenal cortex, testis and perirenal adipose tissue. Serum hormone concentrations were quantified via enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of genes along the HPA and HPG axes were determined using real-time PCR. RESULTS: All types of dietary interventions increased body weight and disturbed metabolic homeostasis, with anxiogenic phenotype in behavioral tests and damage to cell morphology of adrenal cortex and testis being observed. Along the HPA axis, significantly increased corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and corticosterone (CORT) concentrations were observed in the HFD or 0.02% sucralose group. For HPG axis, gonadotropin-releasing hormone (GnRH) and estradiol (E2) concentrations were significantly increased in all dietary intervention groups, while decreased concentrations of follicle-stimulating hormone (FSH) and testosterone (T) were also detected. Moreover, transcriptional profiles of genes involved in the synthesis of hormones and corresponding hormone receptors were significantly altered. CONCLUSION: Long-term consumption of HFD, fructose or sucralose manifested deleterious effects on endocrine system and resulted in the dysregulation of HPA and HPG axes.

De Novo Assembly, Characterization, and Comparative Transcriptome Analysis of Mature Male and Female Gonads of Rabbitfish (Siganus oramin) (Bloch & Schneider, 1801).

The rabbitfish, Siganus oramin, is a commercially important table fish in southeastern China. However, there have been few studies on its gonad development and reproduction regulation. Comparative transcriptome analysis was first performed on adult male and female gonads of S. oramin. In total, 47,070 unigenes were successfully assembled and 22,737 unigenes were successfully annotated. Through comparative transcriptome analysis of male and female gonads, a total of 6722 differentially expressed genes were successfully identified, with 3528 upregulated genes and 3154 downregulated genes in the testes. In addition, 39 differentially expressed reproduction-related genes were identified. Finally, quantitative real-time PCR was used to validate the expression levels of several differentially expressed genes. These results provide important data for further studying the function of reproduction-related genes and the molecular mechanism regulating gonad development and reproduction in S. oramin.

Gonadal Transcriptome Analysis Reveals that SOX17 and CYP26A1 are Involved in Sex Differentiation in the Chinese Soft-Shelled Turtle (Pelodiscus sinensis).

The Chinese soft-shelled turtle (Pelodiscus sinensis) is an important aquaculture animal in China and exhibits growth dimorphism. Single-male cultures are often selected for higher economic efficiency. However, the mechanism of sex differentiation in P. sinensis is not well-known. In this study, a comparative transcriptome analysis of male (ZZ)- and 17ß-oestradiol (E2)-induced pseudo-female (ZZ + E2)-stage embryonic gonads of P. sinensis was performed. A total of 420 differentially expressed genes (DEGs), which included 271 upregulated genes and 149 downregulated genes, were identified. These DEGs were mainly involved in several sex-related pathways, such as "ovarian steroidogenesis", "steroid hormone biosynthesis", "PPAR signalling pathway", and "metabolism of xenobiotics by cytochrome P450". In addition, 50 known and novel candidate genes involved in sex differentiation, such as the male-biased genes AMH, DMRT1, TBX1, and CYP26A1 and the female-biased genes CYP1A1, RASD1, and SOX17, were investigated and identified. For further verification, the full-length cDNAs of SOX17 and CYP26A1 were obtained. SOX17 contains a 1218-bp ORF and encodes 405 amino acids containing an HMG functional domain unique to the Sox superfamily. CYP26A1 contains a 1485-bp ORF and encodes 494 amino acids. Different expression levels of SOX17 and CYP26A1 could be detected in all the tested tissues of males and females. Notably, the expression of CYP26A1 was markedly greater in the gonads of male embryos (P < 0.05) than in those of female embryos, whereas the expression of SOX17 showed the opposite trend (P < 0.05). Taken together, the RNA-seq and qRT‒PCR results suggested potential roles for SOX17 and CYP26A1 in promoting female and male gonadal development, respectively, in P. sinensis. Our results provide new evidence for the mechanism of sex differentiation in P. sinensis.

Active nuclear positioning and actomyosin contractility maintain leader cell integrity during gonadogenesis.

Proper distribution of organelles can play an important role in a moving cell's performance. During C. elegans gonad morphogenesis, the nucleus of the leading distal tip cell (DTC) is always found at the front, yet the significance of this localization is unknown. Here, we identified the molecular mechanism that keeps the nucleus at the front, despite a frictional force that pushes it backward. The Klarsicht/ANC-1/Syne homology (KASH) domain protein UNC-83 links the nucleus to the motor protein kinesin-1 that moves along a polarized acentrosomal microtubule network. Interestingly, disrupting nuclear positioning on its own did not affect gonad morphogenesis. However, reducing actomyosin contractility on top of nuclear mispositioning led to a dramatic phenotype: DTC splitting and gonad bifurcation. Long-term live imaging of the double knockdown revealed that, while the gonad attempted to perform a planned U-turn, the DTC was stretched due to the lagging nucleus until it fragmented into a nucleated cell and an enucleated cytoplast, each leading an independent gonadal arm. Remarkably, the enucleated cytoplast had polarity and invaded, but it could only temporarily support germ cell proliferation. Based on a qualitative biophysical model, we conclude that the leader cell employs two complementary mechanical approaches to preserve its integrity and ensure proper organ morphogenesis while navigating through a complex 3D environment: active nuclear positioning by microtubule motors and actomyosin-driven cortical contractility.

The Rac pathway prevents cell fragmentation in a nonprotrusively migrating leader cell during C. elegans gonad organogenesis.

The C. elegans hermaphrodite distal tip cell (DTC) leads gonadogenesis. Loss-of-function mutations in a C. elegans ortholog of the Rac1 GTPase (ced-10) and its GEF complex (ced-5/DOCK180, ced-2/CrkII, ced-12/ELMO) cause gonad migration defects related to directional sensing; we discovered an additional defect class of gonad bifurcation in these mutants. Using genetic approaches, tissue-specific and whole-body RNAi, and in vivo imaging of endogenously tagged proteins and marked cells, we find that loss of Rac1 or its regulators causes the DTC to fragment as it migrates. Both products of fragmentation-the now-smaller DTC and the membranous patch of cellular material-localize important stem cell niche signaling (LAG-2 ligand) and migration (INA-1/integrin subunit alpha) factors to their membranes, but only one retains the DTC nucleus and therefore the ability to maintain gene expression over time. The enucleate patch can lead a bifurcating branch off the gonad arm that grows through germ cell proliferation. Germ cells in this branch differentiate as the patch loses LAG-2 expression. While the nucleus is surprisingly dispensable for aspects of leader cell function, it is required for stem cell niche activity long term. Prior work found that Rac1-/-;Rac2-/- mouse erythrocytes fragment; in this context, our new findings support the conclusion that maintaining a cohesive but deformable cell is a conserved function of this important cytoskeletal regulator.

Single-nucleus transcriptional and chromatin accessible profiles reveal critical cell types and molecular architecture underlying chicken sex determination.

INTRODUCTION: Understanding the sex determination mechanisms in birds has great significance for the biological sciences and production in the poultry industry. Sex determination in chickens is a complex process that involves fate decisions of supporting cells such as granulosa or Sertoli cells. However, a systematic understanding of the genetic regulation and cell commitment process underlying sex determination in chickens is still lacking. OBJECTIVES: We aimed to dissect the molecular characteristics associated with sex determination in the gonads of chicken embryos. METHODS: Single-nucleus RNA-seq (snRNA-seq) and ATAC-seq (snATAC-seq) analysis were conducted on the gonads of female and male chickens at embryonic day 3.5 (E3.5), E4.5, and E5.5. RESULTS: Here, we provided a time-course transcriptional and chromatin accessible profiling of gonads during chicken sex determination at single-cell resolution. We uncovered differences in cell composition and developmental trajectories between female and male gonads and found that the divergence of transcription and accessibility in gonadal cells first emerged at E5.5. Furthermore, we revealed key cell-type-specific transcription factors (TFs) and regulatory networks that drive lineage commitment. Sex determination signaling pathways, dominated by BMP signaling, are preferentially activated in males during gonadal development. Further pseudotime analysis of the supporting cells indicated that granulosa cells were regulated mainly by the TEAD gene family and that Sertoli cells were driven by the DMRT1 regulons. Cross-species analysis suggested high conservation of both cell types and cell-lineage-specific TFs across the six vertebrates. CONCLUSIONS: Overall, our study will contribute to accelerating the development of sex manipulation technology in the poultry industry and the application of chickens as a unique model for studying cell fate decisions.