Sex Determination Research Articles

GATA4 binding to the Sox9 enhancer mXYSRa/Enh13 is critical for testis differentiation in mouse

In mammals, SOX9/Sox9 expression in embryonic gonads is essential for male gonadal sex determination. Multiple enhancers of Sox9 have been identified, of which the mXYSRa/Enh13 enhancer plays a crucial role in mice. SOX9 and SRY binding sites within the enhancer have been identified as functional. Simultaneous deletion of both sites in mice resulted in male-to-female sex reversal. However, the existence of other critical functional sequences remains unclear. This study identified an additional functional sequence by generating mice with partial deletions in mXYSRa/Enh13. Two nucleotide substitutions within the sequence were sufficient for male-to-female sex reversal. In vivo binding assay by CUT&RUN revealed that GATA4 binds to the sequence. In vitro luciferase assay showed that GATA4 promotes the enhancer activity and the substitution of the sequence reduces the effect. Taken together, the functional sequence in mXYSRa/Enh13 is essential for testis differentiation and requires GATA4 binding.

Male sex determination maintains proteostasis and extends lifespan of daf-18/PTEN deficient C. elegans.

Although females typically have a survival advantage, those with PTEN functional abnormalities face a higher risk of developing tumors than males. However, the differences in how each sex responds to PTEN dysfunction have rarely been studied. We use Caenorhabditis elegans to investigate how male and hermaphrodite worms respond to dysfunction of the PTEN homolog daf-18. Our study reveals that male worms can counterbalance the negative effects of daf-18 deficiency, resulting in longer adult lifespan. The survival advantage depends on the loss of DAF-18 protein phosphatase activity, while its lipid phosphatase activity is dispensable. The deficiency in DAF-18 protein phosphatase activity leads to the failure of dephosphorylation of the endoplasmic reticulum membrane protein C18E9.2/SEC62, causing increased levels of unfolded and aggregated proteins in hermaphrodites. In contrast, males maintain proteostasis through a UNC-23/NEF-mediated protein ubiquitination and degradation process, providing them with a survival advantage. We find that sex determination is a key factor in regulating the differential expression of unc-23 between sexes in response to daf-18 loss. These findings highlight the unique role of the male sex determination pathway in regulating protein degradation.

Isolation and Characterization of GPAT3 Gene from Jojoba Plant and its Inferior Early Diagnosis of Sex.

<b>Background and Objective:</b> In jojoba plants, the sex is usually difficult to identify, especially before flowering and during the very early stages of development. This stage is expected to facilitate breeding programs and adopt an invention and approach to isolate the GPAT gene identified between males and females: The study aimed at early diagnosis of sex in jojoba by sequence characterized by GPAT gene of sex-determining by simplex PCR. To prove the existence of the GPAT gene in male jojoba plants which may be the sex determination and identification in all plant systems. <b>Materials and Methods:</b> Initially, different primers were selected for the sex determination of jojoba samples using PCR-based amplification. The primers that can produce distinct DNA bands in males, not in females were selected for further experiments. The amplification of a male-specific GPAT marker situated in the sex determination region was amplified using specific primers. The newly designed GPAT primers flank region. <b>Results:</b> For the first time, separation and identified of the GPAT gene sequence of jojoba was done. The novel method represents a breakthrough in the sex determination of jojoba to identify sex at early developmental stages. This work provides a potentially useful diagnostic for determining sex in jojoba species. In this report, a breakthrough in the methodology for determining the sex of jojoba has been made. The amplified regions of the GPAT gene closely matched with sequences of GPAT in papaya and humans. <b>Conclusion:</b> The authors make an interesting finding by targeting the sequences in the GPAT gene and the final conclusion that PCR as a simple, rapid and reliable technique can complement and confirm sex by using specific primers pair according to our invention.

Occurrence of Intersex in the Marine Mussel Perumytilus purpuratus (Mollusca: Bivalvia): Does Gonadal Parasitism Play a Role?

Intersexuality is a reproductive phenomenon that occurs in some gonochoric species and refers to the simultaneous presence of both male and female gametes within the same individual. Although this phenomenon has been reported in various invertebrate species, many aspects remain poorly understood, especially in marine mussels. However, it has been suggested that the prevalence of parasites within populations could induce the occurrence of intersex animals. We studied intersexuality in the marine mollusk Perumytilus purpuratus, a key species of the rocky intertidal zone on the southeastern coast of the Pacific Ocean. A total of 6472 mussels from eight locations in northern and southern Chile were analyzed. We estimated the size of the specimens, the sex ratio of the population, and the prevalence of parasites. Additionally, we examined the germ cells of intersex mussels. The results showed that the male-to-female sex ratio (1:1) was maintained in the populations. Intersex mussels were found in six of the eight locations, representing 0.19% of the mussels analyzed. However, no parasites were found in the intersex animals. Additionally, the abundance of intersex individuals was not correlated with parasitism levels in the population. In intersex mussels, the gonadal tissue was compartmentalized, with male and female germ cells remaining separate. It is concluded that intersexuality in Perumytilus purpuratus is a low-frequency reproductive phenomenon, likely resulting from an alteration in the sex determination mechanism. Intersexuality offers a valuable opportunity to explore the biological aspects of sex determination in mussels. Therefore, further research in this area should be pursued.

Effectiveness of prenatal ultrasound in fetal sex identification: a systematic review and meta-analysis.

This systematic review and meta-analysis aimed to assess the accuracy and success rate of ultrasound in determining fetal sex. A search was conducted on Medline, Cochrane Library, and EMBASE databases, and the reference lists of selected studies were also reviewed. Meta-analyses were performed using Revman 5.4.1 and Meta-DiSc 2.0. Twenty-eight studies met the criteria for inclusion in the systematic review. Nine studies reported the accuracy rate of first-trimester fetal sex identification, with a mean accuracy of 85% and a median of 87%. Six studies provided accuracy rates for second and third-trimester identifications, with mean and median rates of 92% and 99%, respectively. A pooled sensitivity and specificity analysis shows that the sensitivity increased from 69% at 11weeks to 89% at 12weeks to 96% at 13weeks. Forest plots on the success rates indicated no significant statistical differences between first-trimester ultrasound sex determination and actual birth sex, with p values of 0.06 for males and 0.08 for females. Similarly, second and third-trimester forest plots showed p values of 0.70 for males and 0.14 for females. In conclusion, ultrasound accuracy for fetal sex determination rises from 87% in first to 99% in second trimesters. The success rate shows no significant difference for either trimester. However, male sex is more easily detected in the second and third trimesters, while female sex is more easily detected in the first trimester. The sensitivity of fetal sex detection in the first trimester increases with gestational age. These findings suggest that prenatal ultrasound sex determination can be useful in managing sex-related pregnancy complications.

Identification of a candidate sex determination region and sex-specific molecular markers based on whole-genome re‑sequencing in the sea star Asterias amurensis.

Sex determination systems are diverse in echinoderms, however, our understanding is still very limited in this research field, especially for Asteroidea species. The northern Pacific seastar, Asterias amurensis, has attracted widespread concern due to its population outbreaks and high-risk invasions. Using whole-genome re-sequencing data from 40 females and 40 males, we identified a candidate sex determination region in A. amurensis. Based on the distribution characteristics of 525 sex-associated single nucleotide polymorphisms, identified by GWAS analysis, 119 sex-specific loci were isolated combining a custom Perl script, PCA analysis and the selection signatures of fixation index FST, suggesting that a 7-12Mb region on chromosome 10 is a candidate sex-determining region. The existence of female-specific sequences and the genotypes of sex-specific loci indicated that A. amurensis might utilize a ZZ/ZW sex determination system. We also developed two pairs of sex-specific primers which could distinguish the genetic sex of this starfish with 100% accuracy. As the first study on sex determination in Asteroidea, it will provide novel insights into diverse sex determination systems in echinoderms and allow for in-depth studies on sex-related eco-physiological issues in A. amurensis.

Sex Determination in Reptiles: A Review

Sex Determination in Reptiles: A Review

Antenatal Diagnosis and Treatment in Congenital Adrenal Hyperplasia Due to 21-hydroxylase Deficiency and Congenital Adrenal Hyperplasia Screening in Newborns

Signs of virilization, such as clitoromegaly, labio-scrotal fusion, and urogenital sinus may be observed in females with 21-hydroxylase deficiency (21-OHD) and other rare virilizing forms of congenital adrenal hyperplasia (CAH). This makes sex determination difficult, and multiple reconstructive surgeries in the postnatal period may be required. As 21-OHD is an autosomal recessive disease, the chance of any child being affected is one in four and so only one in eight will be an affected female. The primary objective of antenatal diagnosis is to identify only the affected fetus in the early gestational weeks before the onset of genital organogenesis and to treat that case. Therefore, studies aimed at antenatal diagnosis and preventing adrenal androgen exposure in the female fetus with CAH have long been of interest. Antenatal steroid treatment is considered experimental and controversial for safety reasons in recent clinical guidelines. If antenatal treatment is to be used, it is recommended that it should be performed in experienced centers that can collect data on a large number of cases which will help to define the benefits and harms of treatment better. In the postnatal period, a severe deficiency of the 21-hydroxylase enzyme leads to life-threatening adrenocortical insufficiency in both sexes and varying degrees of pathology of the external genitalia in females. This condition is also associated with high mortality in the first days of life and an increased risk of incorrect sex assignment. Neonatal screening for 21-OHD CAH effectively detects the severe forms and reduces mortality, and it is instrumental in the correct sex assignment of female cases.

The master male sex determinant Gdf6Y of the turquoise killifish arose through allelic neofunctionalization

Although sex determination is a fundamental process in vertebrate development, it is very plastic. Diverse genes became major sex determinants in teleost fishes. Deciphering how individual sex-determining genes orchestrate sex determination can reveal new actors in sexual development. Here, we demonstrate that the Y-chromosomal copy of the TGF-β family member gdf6 (gdf6Y) in Nothobranchius furzeri, an emerging model organism in aging research, gained the function of the male sex determinant through allelic diversification while retaining the skeletal developmental function shared with the X-chromosomal gdf6 allele (gdf6X). Concerning sex determination, gdf6Y is expressed by somatic supporting cells of the developing testes. There it induces the male sex in a germ cell-independent manner in contrast to sex determination in zebrafish and the medaka. Looking for downstream effectors of Gdf6Y, we identified besides TGF-β signaling modulators, especially the inhibitor of DNA binding genes id1/2/3, the mRNA decay activator zfp36l2 as a new GDF6 signaling target.

Missense variants weakening a SOX9 phosphodegron linked to odontogenesis defects, scoliosis and other skeletal features.

SOX9 encodes an SRY-related transcription factor critical for chondrogenesis and sex determination among other processes. Loss-of-function variants cause campomelic dysplasia and Pierre Robin Sequence, while both gain- and loss-of-function variants cause disorders of sex development. SOX9 has also been linked to scoliosis and cancers, but variants are undetermined. It is highly expressed in tooth progenitor cells, but its odontogenic roles remain elusive and tooth defects are unreported in SOX9-related conditions. We here performed whole-exome sequencing for nine unrelated children with tooth eruption delay and no known syndromes, and identified a seven-year-old girl heterozygous for a SOX9 p.Thr239Pro variant and a ten-year-old boy heterozygous for presumably adjacent p.Thr239Pro and p.Thr240Pro variants. These variants were de novo and rare in control populations. Both cases had primary tooth eruption delay. Additionally, the boy had mesiodens blocking permanent central upper incisor eruption, severe scoliosis and mild craniofacial and appendicular skeleton abnormalities. p.Thr239 and p.Thr240 occupy variable and obligatory positions, respectively, in a Cdc4/FBXW7-targeted phosphodegron motif fully conserved in SOX9 vertebrate orthologs and SOX8 and SOX10 paralogs, but functionally uncharacterized in vivo. Structural modeling predicted p.Thr240Pro and p.Thr239Pro/p.Thr240Pro but not p.Thr239Pro to strongly reduce SOX9/FBXW7 interaction. Accordingly, p.Thr240Pro and p.Thr239Pro/p.Thr240Pro but not p.Thr239Pro blocked FBXW7-induced SOX9 degradation in cultured cells. All variants increased SOX9-mediated reporter activation independently of protein stabilization, suggesting that CPD may also modulate SOX9's transactivation function. Altogether, these findings concur that CPD has critical functions, that SOX9 decisively controls odontogenesis, and that gain-of-function variants may markedly perturb both this process and skeletogenesis.

CRISPR targeting of FOXL2 c.402C>G mutation reduces malignant phenotype in granulosa tumor cells and identifies anti-tumoral compounds.

Forkhead box L2 (FOXL2) encodes a transcription factor essential for sex determination, and ovary development and maintenance. Mutations in this gene are implicated in syndromes involving premature ovarian failure and granulosa cell tumors (GCTs). This rare cancer accounts for less than 5% of diagnosed ovarian cancers and is causally associated with the FOXL2 c.402C>G, p.C134W mutation in 97% of the adult cases (AGCTs). In this study, we employed CRISPR technology to specifically eliminate the FOXL2 c.402C>G mutation in granulosa tumor cells. Our results show that this Cas9-mediated strategy selectively targets the mutation without affecting the wild-type allele. Granulosa cells lacking FOXL2 c.402C>G exhibit a reduced malignant phenotype, with significant changes in cell proliferation and invasion. Furthermore, these modified cells are more susceptible to dasatinib and ketoconazole. Transcriptomic and proteomic analyses reveal that CRISPR-modified granulosa tumor cells shift their expression profiles towards a wild-type-like phenotype. Additionally, this altered expression signature has led to the identification of new compounds with antiproliferative and pro-apoptotic effects on granulosa tumor cells. Our findings demonstrate the potential of CRISPR technology for the specific targeting and elimination of a mutation causing GCTs, highlighting its therapeutic promise for treating this rare ovarian cancer.

Lactoferrin Attenuates Pro-Inflammatory Response and Promotes the Conversion into Neuronal Lineages in the Astrocytes.

Neurodegenerative diseases are characterized by progressive loss of neurons and persistent inflammation. Neurons are terminally differentiated cells, and lost neurons cannot be replaced since neurogenesis is restricted to only two neurogenic niches in the adult brain, whose neurogenic potential decreases with age. In this regard, the astrocytes reprogramming into neurons may represent a promising strategy for restoring the lost neurons and rebuilding neural circuits. To date, many anti-inflammatory agents have been shown to reduce neuroinflammation; however, their potential to restore neuronal loss was poorly investigated. This study investigates the anti-inflammatory effects of lactoferrin on DI-TNC1 astrocyte cell line and its ability to induce astrocyte reprogramming in a context of sustained inflammation. For this purpose, astrocytes were pre-treated with lactoferrin (4 μg/mL) for 24 h, then with lipopolysaccharide (LPS) (400 ng/mL), and examined 2, 9 and 16 days from treatment. The results demonstrate that lactoferrin attenuates astrocyte reactivity by reducing Toll-like receptor 4 (TLR4), Glial fibrillary acidic protein (GFAP) and IL-6 expression, as well as by upregulating Interleukin-10 (IL-10) cytokine and NRF2 expression. Moreover, lactoferrin promotes the reprogramming of reactive astrocytes into proliferative neuroblasts by inducing the overexpression of the Sex determining region Y/SRY-box 2 (SOX2) reprogramming transcription factor. Overall, this study highlights the potential effects of lactoferrin to attenuate neuroinflammation and improve neurogenesis, suggesting a future strategy for the treatment of neurodegenerative disorders.

Developmental pathways underlying sexual differentiation in the U/V sex chromosome system of giant kelp.

In many multicellular organisms, sexual development is not determined by XX/XY or ZW/ZZ systems but by U/V sex chromosomes. In U/V systems, sex determination occurs in the haploid phase, with U chromosomes in females and V chromosomes in males. Here, we explore several male, female, and partially sex-reversed male lines of giant kelp to decipher how U/V sex chromosomes and autosomes initiate male versus female development. We identify a key set of genes on the sex chromosomes involved in triggering sexual development and characterize autosomal effector genes underlying sexual differentiation. We show that male, but not female, development involves large-scale transcriptome reorganization with pervasive enrichment in regulatory genes, faster evolutionary rates, and high species-specificity of male-biased genes. Our observations imply that a female-like phenotype is the "ground state", which is complemented by the presence of aU-chromosome but overridden by a dominant male developmental program triggered by the V-chromosome.

Comparative analysis for sexual dimorphism of mandibular canine in two ethnic diverse population with clinical and experimental method

Background: Sex determination is challenging with minimal skeletal remains. It is overcome by odontometric measurement of mandibular canines as it exhibits higher sexual dimorphism in the human dentition. Hence, the study aimed to assess and validate mandibular canine in gender determination using odontometric measurements in two ethnically diverse populations. Methods: A total of 205 subjects, belonging to two ethnically diverse population, i.e., Indian (Himachali) and Nepali, aged 17–25 years was included. Intraoral measurement outcome comprised both-sided lower mandibular canine distance including intercanine distance using Vernier caliper, followed by similar measurements on study models (casts). Garns and Lens formula was utilized to assess the sexual dimorphism. Results: The canine distance of both sides in Nepali males was higher than Indian males, both intraoral and casts. The intercanine distance in Indian males was higher than Nepali males, both intraoral and casts. The canine and intercanine distance of both sides was greater in Indian females than Nepali females, both intraoral and casts. Indian population in the left-sided lower canine had higher sexual dimorphism than the right-sided lower canine both intraorally and casts in contrast to Nepali population that exhibited higher in the right-sided canine. Conclusion: Therefore, mandibular canine measurements were an adjunct and reliable method for gender identification in human.

Functions of the transcription factor myb-like in sex determination and differentiation in Portunus trituberculatus

Functions of the transcription factor myb-like in sex determination and differentiation in Portunus trituberculatus

Gonadal expression profiles reveal the underlying mechanisms of temperature effects on sex determination in the large-scale loach (Paramisgurnus dabryanus)

The sex determination mechanism in large-scale loach (Paramisgurnus dabryanus) follows a ZZ/ZW system, with sexual differentiation regulated by both genotypic factors and temperature effects (GSD+TSD), where elevated temperatures result in a higher proportion of males. Currently, research on the sex determination mechanisms in large-scale loach is limited, and the specific gene expression profiles and the role of temperature in influencing sex remain largely unknown. This study investigated the impact of temperature on the sex ratio in cultured populations of the large-scale loach, and then identified a female-specific genetic marker by whole genome sequencing, facilitating the distinguishing of females, males, and pseudo-males within this population. Transcriptomic analysis was subsequently performed on these groups, and the data revealed a similar expression pattern between pseudo-males and true-males. The research combined differential expression analysis with WGCNA to construct a regulatory network of nine sex differentiation-related genes (SDG) (map3k4, trpv4, hsd17b12a, wt1, ar, dmrt1, bcar1, sox9a, cyp17a1), indicating that sex differentiation in large-scale loach is probably driven by the regulation of male-related genes. The transcriptomic analysis suggested that temperature significantly modified the expression of SDG in the ovaries, while in the testes, it predominantly affects metabolism-related pathways. We established a temperature-sensitive gene network in females, based on the correlation between gene expression and temperature, as well as the number of co-regulated genes in female data. We propose that, with increasing temperature, wt1 serves as a central regulator, leading to the down-regulation of foxl2a, cyp19a1a, and the cholesterol biosynthesis-related gene sqlea, ultimately resulting in the development of pseudo-males.

Egg Characteristics Assessment as an Enabler for In-ovo Sexing Technology: a review

The culling of day-old male chicks remains a significant challenge for the egg-laying industry. While in ovo sexing technology has made strides, it has yet to achieve an optimal balance between early identification of sex at hatching, high sensitivity, and non-invasive operation. This paper aims to provide a comprehensive review of the research progress in in ovo sexing from various perspectives and to explore potential solutions in light of industrial practices and key technological bottlenecks. This study describes an efficient detection method for low-concentration samples and provides recommendations for the rapid detection of sex markers in chicken embryo allantoic fluid. Given the complexity of optical signals and the significant individual differences among eggs, the paper underscores the importance of evaluating egg characteristics in advancing in ovo sex determination technology. Based on this core concept, the use of multimodal data fusion strategies are advocated for in ovo sexing. This approach aims to deepen researchers' understanding of the complex relationship between sex labels and the high-dimensional data of breeding eggs, thereby injecting new momentum and direction into the development of this field.

Sex determination factor, a novel male-linked gene in the sea cucumber Apostichopus japonicus: Molecular characterization, expression patterns and effects of gene knockdown.

Apostichopus japonicus is a highly significant marine aquaculture species. Research findings have indicated that male sea cucumbers demonstrate a more rapid growth rate compared to females, underscoring the potential advantages of establishing an all-male population. In this study, we identified a specific protein-coding gene (ORFan) within a 4565bp male fragment and named it sex determination factor (sdf). The sdf transcript exhibited ubiquitous expression in various adult male tissues, along with dynamic expression patterns in the testis across different developmental stages. Notably, knockdown of the sdf gene through immersion of embryos in its specific vivo-morpholino oligomers (vivo-MO) resulted in significant changes in the expression levels of several sex-related genes including piwi1, vasa, foxl2, and DNMT3. Additionally, a transcriptomic analysis showed that sdf knockdown resulted in significant alterations in multiple biological processes encompassing various sex-related gene ontology terms such as male gonad development, ovarian follicle development, and steroidogenesis. These results provide a molecular foundation for comprehending ORFans in sea cucumbers while offering a valuable method for gene knockdown studies in echinoderms.

Gender-based Morphometric Analysis of the Human Orbital Aperture Profile using 3D Reconstruction Computed Tomography: A Cross-sectional Retrospective Study

Introduction: Sex estimation is a crucial aspect of forensic medicine and requires primary consideration when examining skeletal remains. Among the various anatomical features, the orbital region holds significant importance due to its distinct morphological variations. These variations provide valuable insights for sex determination, making orbital anatomy an indispensable tool in forensic investigations and analysis. Aim: To measure and compare orbital apertures between males and females using 3D Computed Tomography (CT) in a sample from the Saudi population. Materials and Methods: A cross-sectional retrospective study was conducted from January to June 2024 in the Radiology Department of Tower Hospital, Arar, Kingdom of Saudi Arabia, following the acquisition of ethical approval from the Local Bioethics Committee at Northern Border University. A total of 100 CT scans were randomly selected from the available dataset. The study encompassed CT scans of subjects from both genders, aged 18 years and above. However, CT scans of patients who had sustained head trauma or orbital injuries were excluded from the study. The data retrieved were analysed using Statistical Package for the Social Sciences (SPSS) version 23.0. All continuous data were tested for normality and expressed as Mean±Standard deviation (SD). The significant differences in orbital measurements between males and females were assessed using the Independent t-test. A p-value of <0.05 was considered statistically significant. Results: A total of 100 participants were included in the study, with the majority being females (52%). The mean age of male and female subjects was 40.5±13.19 years and 39.13±13.10 years, respectively. A statistically significant difference was found in right orbital width (p-value=0.011), left orbital area (p-value=0.04), and Interzygomatic Distance (IZD), with these measurements being higher in males compared to females (p-value=0.009). Conclusion: The study revealed that males have greater orbital width, orbital area and IZD compared to females. These genderspecific variations in orbital and facial dimensions may play a crucial role in gender determination within forensic medicine.

Is Imane Khelif Male or Female

The Summer Olympic games of 2024 saw a peculiar case of uncertain sex determination, specifically in the women’s boxing competition. Algerian boxer Imane Khelif’s eligibility to compete was brought into question. Khelif had previously been banned from the women’s World Boxing Championship due to a failure to pass their standards for female athletes. Essentially, the International Boxing Association ruled that Khelif was not female. The Olympic Committee ruled otherwise and Khelif eventually won gold in the competition, causing significant controversy along the way. In this article, Jill Cook examines the question of whether Khelif is male or female by examining several disorders in sexual development that may have contributed to the confusion surrounding Khelif’s sex.