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Coexistence of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency and Turner Syndrome (TS) is rare. We report a 6-year-old Portuguese girl with mosaic TS [45,XO(39)/47,XXX(21)] presented with premature pubarche at the age of 5 years. Laboratory findings showed elevated 17-hydroxyprogesterone, dehydroepiandrosterone sulfate, androstenedione and total testosterone, and sex-determining region Y (SRY) was negative. CYP21A2 gene analysis revealed two mutations (c.[844G>T]; [CYP21A2del]), consistent with the non-classical form of CAH. Complete deletion of CYP21A2 allele occurred de novo. At 6 years and 4 months, she presented accelerated growth velocity and hydrocortisone at the dose of 5 mg/m2/day was initiated. This case highlights the need to perform global examinations looking for virilization signs in TS patients follow-up. It also supports the reported genetic combination of TS and CAH. Therefore, CAH should be kept in mind in TS patients with SRY negative and virilization signs, even in the absence of short stature.

This chapter begins with a discussion on sex determination. The sex of an individual is the outcome of genetic determinants and of hormonal effects that confer structural features characteristic of a given sex. Both genetic and hormonal determinants normally operate at only two phases of life: during fetal development and at puberty. After about eight weeks of gestation, the product of a specific gene called sex-determining region Y (SRY) in the Y-chromosome inhibits the action of dosage-sensitive sex reversal, adrenal hypoplasia congenital, critical region on the X chromosome, gene 1(DAX-1), a gene located in the X-chromosome that results in the development of testes. In the absence of the Y-chromosome, the X-chromosome directs the development of fetal gonads into ovaries. The chapter further examines the regulation of the reproductive system in females and males. This is determined by external environmental cues and the interaction between internal signals located in specific brain centers, the hypothalamus, anterior pituitary, gonads, and peripheral tissues. These interrelationships are commonly known as the hypothalamic-pituitary-gonadal axis and are integrated and programmed by several hormones.

The Trachinotus ovatus is a popular aquaculture species in China. There are no obvious morphological differences between male and female fish, even during maturity, prompting research studies on sex-related features of this fish. To examine sex determination- and gonadal development-related genes, we conducted transcriptome analysis of the ovaries and testes of T. ovatus. A total of 345,972,132 high-quality clean reads were obtained from 12 libraries. In addition, 28,137 gonad-expressed unigenes were obtained by mapping the clean reads to the T. ovatus reference genome. A total of 8,237 differentially expressed genes (DEGs) were identified between stage I ovaries and testes, including 3,235 testicular upregulated and 5,002 ovarian upregulated genes. Furthermore, 13,448 DEGs were obtained between stage III ovarian and testicular libraries, including 7,576 testicular upregulated and 5,872 ovarian upregulated genes. The DEGs included some sex-determining genes such as sry, dmrt1, and amh. DEGs between ovarian and testicular libraries were significantly enriched with KEGG pathways that are involved in gonadal development, sex determination, and gonadal function. Then, 10 DEGs (seven testicular upregulated and three ovarian upregulated unigenes) were selected for quantitative real-time PCR analysis. Four genes (zinc-binding protein A33-like, pro-opiomelanocortin-like, leucine-rich repeat and transmembrane domain-containing protein 2-like, and forkhead boxC1) showed a specific testicular expression pattern. The gonadally expressed as well as testicular and ovarian DEGs provide useful information for further research on the reproductive biology of T. ovatus.

Turner Syndrome (TS) is defined as a total or partial loss of the second sex chromosome in a phenotypically female patient. Due to the possibility of hidden mosaicism of fragments of the Y chromosome and the development of gonadoblastoma, we proposed in this study to evaluate the presence of these fragments in two tissues with different embryonic origin, peripheral blood lymphocytes (mesoderm) and oral mucosa cells (ectoderm) by the technique multiplex PCR. DNA samples were collected from 109 patients and primers for the SRY, TSPY and AMELX genes were used. We found 14 patients (12.8%) with positive molecular markers for Y chromosome. The study of tissues of different embryological origin, such as blood and oral tissue obtained the same degree of agreement, as well as the same sensitivity and specificity. The use of oral mucosa cells is a simpler method of collection and less invasive, has less time for DNA extraction at a lower cost.

Without known analogous sex-determining factors like SRY (sex determining region Y) in mammals, the chicken (Gallus gallus) sex determination mechanism still remains unclear, which highly restricts the biological research on chicken development and poultry single-sex reproduction. Here we not only characterized a new female-biased gene UBE2I and identified the expression pattern by qRT-PCR, but also described the functional role of UBE2I in the gonadal development of chicken embryos. Results showed that UBE2I exhibited a female-biased expression pattern in the early stage of PGCs (primordial germ cells) in embryonic gonads and robust expression in ovaries of newborn chickens. Most importantly, we successfully developed an effective method to interfere or overexpress UBE2I in chicken embryos through the intravascular injection. The qRT-PCR analysis showed that the sex-related genes (FOXL2, CYP19A1 and HINTW) in females were upregulated (P

SOX10 belongs to a family of 20 SRY (sex-determining region Y)-related high mobility group box-containing (SOX) proteins, most of which contribute to cell type specification and differentiation of various lineages. The first clue that SOX10 is essential for development, especially in the neural crest, came with the discovery that heterozygous mutations occurring within and around SOX10 cause Waardenburg syndrome type 4. Since then, heterozygous mutations have been reported in Waardenburg syndrome type 2 (Waardenburg syndrome type without Hirschsprung disease), PCWH or PCW (peripheral demyelinating neuropathy, central dysmyelination, Waardenburg syndrome, with or without Hirschsprung disease), intestinal manifestations beyond Hirschsprung (ie, chronic intestinal pseudo-obstruction), Kallmann syndrome and cancer. All of these diseases are consistent with the regulatory role of SOX10 in various neural crest derivatives (melanocytes, the enteric nervous system, Schwann cells and olfactory ensheathing cells) and extraneural crest tissues (inner ear, oligodendrocytes). The recent evolution of medical practice in constitutional genetics has led to the identification of SOX10 variants in atypical contexts, such as isolated hearing loss or neurodevelopmental disorders, making them more difficult to classify in the absence of both a typical phenotype and specific expertise. Here, we report novel mutations and review those that have already been published and their functional consequences, along with current understanding of SOX10 function in the affected cell types identified through in vivo and in vitro models. We also discuss research options to increase our understanding of the origin of the observed phenotypic variability and improve the diagnosis and medical care of affected patients.

The molecular background of disorders of sex development (DSD) in dogs is poorly understood. Several copies of the SRY genes have been reported in the dog genome. We used droplet digital PCR with the aim of determining variability in SRY copy number and its association with DSD in dogs. Altogether 19 DSD male dogs (XY DSD) of 10 breeds and 87 control dogs of eight breeds were analyzed. Moreover, we performed a comparative analysis of SRY copy number in other canids: wolves (3), red foxes (16), and Chinese raccoon dogs (10). We found that the modal number of SRY copies in dogs, wolves, red foxes, and Chinese raccoon dogs was 3, 3, 1, and 3 respectively. Variability of copy number was only observed in Yorkshire Terriers (two or three copies) and red foxes (one or two copies). An analysis of six DSD Yorkshire Terriers and 38 control males of this breed showed that 50% of the DSD dogs had two copies, while the incidence of this variant was significantly lower in the control dogs (10.5%). Searching for the copy number of the coding and 5'-flanking fragments revealed full concordance with the copy number. These fragments were also sequenced in DSD (19) and control (24) dogs, and no DNA variants were found. We conclude that, in the dog, two or three functional copies of the SRY gene are present, and a smaller number of copies showed an association with the risk of DSD phenotype in Yorkshire Terriers.

Intrahepatic cholangiocarcinoma (ICC) develops rapidly with a high malignancy. SOX9 expression is increased in several tumors. However, its expression and role in intrahepatic cholangiocarcinoma have not yet been elucidated. Real time PCR and Western blot were done to assess SOX9 expression in tumor tissues and adjacent tissues of ICC. ICC cell line QBC939 cells were separated into control group, SOX9 overexpression group and SOX9 siRNA group followed by analysis of cell survival by MTT assay, cell migration by cell scratch assay, cell invasion by transwell chamber, E-cadherin and Vimentin level by western blot, TGFβ/Smad signaling protein level by real time PCR. SOX9 level in tumor tissues was significantly increased compared to adjacent tissues (P < 0.05) and it was associated with TNM stage, tissue type and metastasis, and survival time (P < 0.05). Transfection of pcDNA3.1-SOX9 upregulated SOX9, promoted cell proliferation, migration and invasion, downregulated E-cadherin, upregulated Vimentin, TGF-β1 and Smad4 (P < 0.05). SOX9 siRNA transfection into QBC939 cells could significantly reverse the above mentioned changes (P < 0.05). SOX9 level is increased in intrahepatic cholangiocarcinoma and targeting SOX9 can inhibit cell migration and invasion, and EMT via regulating TGFβ/Smad signaling.

The use of mesenchymal stem cells can add local improvements potential to enthesis tissue regeneration based on tropical activity through secretions of growth factors, cytokines, and vesicles (e.g. exosomes), collectively known as secretomes. This study aims to analyze secretomes characterization from adipose-derived mesenchymal stem cells seeded with enthesis tissue scaffold in hypoxic conditions and to analyze the influence of hypoxic environment to the characterization of secretomes. This is an in-vitro study using a Randomized Control Group Post-Test Only design. This study using Adipose Stem Cells (ASCs) were cultured in hypoxia (Oxygen 5%) and Normoxia (21%) condition. The scaffolds are fresh-frozen enthesis tissue and was seeded in the treatment group and compared to control. The evaluation of Scleraxis (Scx) and SRY-box (Sox9) was measured using ELISA on the 2nd, 4th, and 6th days. Comparison of Scx levels between each evaluation time showed a positive trend in a group with scaffold in hypoxia condition although it has no significant differences (p=0.085), with the highest level on day 6, that is 13,568 ng/ml. Conversely, the comparison of Sox9 showed significant differences (p=0.02) in a group with scaffold in hypoxia condition, with the highest level on day 4, that is 28,250 ng/ml. The use of enthesis scaffold seeded in adipose-derived mesenchymal stem cells in hypoxic conditions shows a positive trend as regenerative effort of injured enthesis tissue through Scleraxis and Sox9 secretomes induction.

The study aimed to determine the variation of Y-chromosome-bearing sperm content among individual ejaculates. A real-time polymerase chain reaction (qPCR) with unique primers was developed and used to calculate the percentage of Y-chromosome-bearing sperm in individual ejaculates from 50 randomly selected men. There was a significant difference in the overall mean ± SD between the proportion of Y-chromosome-bearing sperm and X-chromosome-bearing sperm (45.36 ± 7.88 vs. 54.42 ± 7.88). Of the 50 ejaculates, 17 had more than, and 14 had less than the 99% confidence interval of the mean of the Y-chromosome-bearing sperm (45.58 ± 2.87). These results suggest that the inconsistency in sperm-based sex-selection outcomes appears to be a function of differences in the ejaculates and highlights the need for further study in environmental and genetic factors contributing to X or Y bearing spermatozoan instability.Abbreviations: qPCR: real-time polymerase chain reaction; ROS: reactive oxygen species; DTT: dithiothreitol; SRY: sex-determining region Y.

Sexual dimorphism in gene regulation, including DNA methylation, is the main driver of sexual dimorphism in phenotypes. However, the questions of how and when sex shapes DNA methylation remain unresolved. Recently, using mice with different combinations of genetic and phenotypic sex, we identified sex-associated differentially methylated regions (sDMRs) that depended on the sex phenotype. Focusing on a panel of validated sex-phenotype dependent male- and female-biased sDMRs, we tested the developmental dynamics of sex bias in liver methylation and the impacts of mutations in the androgen receptor, estrogen receptor alpha, or the transcriptional repressor Bcl6 gene. True hermaphrodites that carry both unilateral ovaries and contralateral testes were also tested. Our data show that sex bias in methylation either coincides with or follows sex bias in the expression of sDMR-proximal genes, suggesting that sex bias in gene expression may be required for demethylation at certain sDMRs. Global ablation of AR, ESR1, or a liver-specific loss of BCL6, all alter sDMR methylation, whereas presence of both an ovary and a testis delays the establishment of male-type methylation levels in hermaphrodites. Moreover, the Bcl6-LKO shows dissociation between expression and methylation, suggesting a distinct role of BCL6 in demethylation of intragenic sDMRs.

A diverse array of sex determination ( SD) mechanisms, encompassing environmental to genetic, have been found to exist among vertebrates, covering a spectrum from fixed SD mechanisms (mammals) to functional sex change in fishes (sequential hermaphroditic fishes). A major landmark in vertebrate SD was the discovery of the SRY gene in 1990. Since that time, many attempts to clone an SRY ortholog from nonmammalian vertebrates remained unsuccessful, until 2002, when DMY/dmrt1by was discovered as the SD gene of a small fish, medaka. Surprisingly, however, DMY/dmrt1by was found in only 2 species among more than 20 species of medaka, suggesting a large diversity of SD genes among vertebrates. Considerable progress has been made over the last 3 decades, such that it is now possible to formulate reasonable paradigms of how SD and gonadal sex differentiation may work in some model vertebrate species. This review outlines our current understanding of vertebrate SD and gonadal sex differentiation, with a focus on the molecular and cellular mechanisms involved. An impressive number of genes and factors have been discovered that play important roles in testicular and ovarian differentiation. An antagonism between the male and female pathway genes exists in gonads during both sex differentiation and, surprisingly, even as adults, suggesting that, in addition to sex-changing fishes, gonochoristic vertebrates including mice maintain some degree of gonadal sexual plasticity into adulthood. Importantly, a review of various SD mechanisms among vertebrates suggests that this is the ideal biological event that can make us understand the evolutionary conundrums underlying speciation and species diversity.

Background Male sex reversal syndrome is a rare genetic cause of male infertility with an overall incidence of 1/20,000–1/100,000 males. There is mismatching between the genetic make-up and the apparent clinical features. The clinical presentation of such cases is variable ranging from ambiguous genitalia at birth, failed puberty, up to normal male phenotype with infertility and hypogonadism. The exact molecular and genetic bases of this syndrome are still unclear. Most of the recorded cases were SRY positive (i.e. representing 80–90% of all cases), and they showed translocated SRY gene on the Y chromosome. Moreover, fewer cases of male sex reversal (46, XX) were SRY negative. Case presentation Herby, we report a rare case of a 35-year-old infertile male patient who presented with azoospermia, hypergonadotropic hypogonadism, and abnormal classical (46, XX) karyotype, as well as negative FISH for SRY gene. He had a previous negative biopsy and was asking for redoing micro-TESE, whoever he was discouraged as chances to find sperm is eventually nil, and instead, he was prescribed testosterone replacement therapy to correct hypogonadism. Conclusion Therefore, any case of non-obstructive azoospermia should be offered genetic testing trying to exclude non-treatable cases and for genetic counseling.

Background Constitutional telomeric associations are very rare events and the mechanism underlying their development is not well understood. Case presentation We here describe a female case of Turner syndrome with a 45,X,add(22)(p11.2)[25]/45,X[5]. We reconfirmed this karyotype by FISH analysis as 45,X,dic(Y;22)(p11.3;p11.2)[28]/45,X[2].ish dic(Y;22)(SRY+,DYZ1+). A possible mechanism underlying this mosaicism was a loss of dic(Y;22) followed by a monosomy rescue of chromosome 22. However, SNP microarray analysis revealed no loss of heterozygosity (LOH) in chromosome 22, although a mosaic pattern of LOH was clearly detectable at the pseudoautosomal regions of the sex chromosomes. Conclusions Our results suggest that the separation of the dicentric chromosome at the junction resulted in a loss of chromosome Y without a loss of chromosome 22, leading to this patient’s unique mosaicism. Although telomere signals were not detected by FISH at the junction, it is likely that the original dic(Y;22) chromosome was generated by unstable telomeric associations. We propose a novel “pulled apart” mechanism as the process underlying this mosaicism.

Background/aim: Prenatal diagnosis is vital to obtain healthy generation for risky pregnancies. There have been several approaches, some of which are routinely applied in clinics to evaluate the possible prenatal deficiencies and/or diseases. In the present study, we aimed to isolate the fetal cells from endocervical samples and try to identify possible anomalies which were proved by Amniocentesis (AS) and chorionic villus sampling (CVS) methods. Materials and methods: Endoservical specimens were collected from 100 pregnant women. Cells were separated in parallel by fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) using human leukocyte antigen (HLA) G233 and placental alkaline phosphatase (PLAP) antibodies. CMA (comprehensive meta-analysis) were carried out and male fetuses were confirmed with Sex determining region Y (SRY) amplification. Results: The percent of HLA G233 and placental and placental alkaline phosphatase (PLAP) positive cells were 4.55% and 84.59%, respectively. The percent of cells positive for both markers was 14.75%. CMA analyses were not informative. (SRY) was amplified in 67% of the samples. Conclusion: However, the success rate of the both cell sorting and scanning of DNA anomalies by aCGH and/or RT-PCR was limited, preventing the applicability of this proposal in the clinics. Still, the success of the proposed method depends on the development of the novel fetal cell-specific antibodies and the improvements in the sorting systems.

Testis differentiation is initiated when Sry in pre-Sertoli cells directs the gonad toward a male-specific fate. Sertoli cells are essential for testis development, but cell types within the interstitial compartment, such as immune and endothelial cells, are also critical for organ formation. Our previous work implicated macrophages in fetal testis morphogenesis, but little is known about genes underlying immune cell development during organogenesis. Here we examine the role of the immune-associated genes Mafb and Maf in mouse fetal gonad development, and we demonstrate that deletion of these genes leads to aberrant hematopoiesis manifested by supernumerary gonadal monocytes. Mafb;Maf double knockout embryos underwent initial gonadal sex determination normally, but exhibited testicular hypervascularization, testis cord formation defects, Leydig cell deficit, and a reduced number of germ cells. In general, Mafb and Maf alone were dispensable for gonad development; however, when both genes were deleted, we observed significant defects in testicular morphogenesis, indicating that Mafb and Maf work redundantly during testis differentiation. These results demonstrate previously unappreciated roles for Mafb and Maf in immune and vascular development and highlight the importance of interstitial cells in gonadal differentiation.Summary statementDeletion of Mafb and Maf genes leads to supernumerary monocytes in fetal mouse gonads, resulting in vascular, morphogenetic, and differentiation defects during testicular organogenesis.

The mammalian sex chromosome system (XX female/XY male) is ancient and highly conserved. The sex chromosome karyotype of the creeping vole (Microtus oregoni) represents a long-standing anomaly, with an X chromosome that is unpaired in females (X0) and exclusively maternally transmitted. We produced a highly contiguous male genome assembly, together with short-read genomes and transcriptomes for both sexes. We show that M. oregoni has lost an independently segregating Y chromosome and that the male-specific sex chromosome is a second X chromosome that is largely homologous to the maternally transmitted X. Both maternally inherited and male-specific sex chromosomes carry fragments of the ancestral Y chromosome. Consequences of this recently transformed sex chromosome system include Y-like degeneration and gene amplification on the male-specific X, expression of ancestral Y-linked genes in females, and X inactivation of the male-specific chromosome in male somatic cells. The genome of M. oregoni elucidates the processes that shape the gene content and dosage of mammalian sex chromosomes and exemplifies a rare case of plasticity in an ancient sex chromosome system.

Functional elucidation of bovine Y-chromosome genes requires available genome editing technologies. Meanwhile, it has yet to be proven whether the bovine Sry gene is the main or single factor involved in the development of the male phenotype in bovine. Here, we efficiently knocked out four Y-linked genes (Sry, ZFY, DDX3Y, and EIF2S3Y) in bovine fetal fibroblasts (BFFs) with transcription activator-like effector nucleases (TALENs) individually. Furthermore, we used TALEN-mediated gene knockin at the Sry gene and generated a sex-reversal bovine by somatic cell nuclear transfer (SCNT). The resulting bovine had only one ovary and was sterile. We demonstrate, for the first time, that the Sry gene is an important sex-determining gene in bovine. Our method lays a solid foundation for detecting the biology of the bovine Y chromosome, as it may provide an alternative biological model system for the study of mammalian sex determination, and new methods for the practical application in agricultural, especially for sex predetermination.

Sex determination requires the commitment of bipotential gonads to either a testis or an ovarian fate. Gene deletion of the kinase Map3k4 results in gonadal sex reversal in XY mice, and transgenic re-expression of Map3k4 rescues the sex reversal phenotype. Map3k4 encodes a large, multi-functional protein possessing a kinase domain and several, additional protein–protein interaction domains. Although MAP3K4 plays a critical role in male gonadal sex determination, it is unknown if the kinase activity of MAP3K4 is required. Here, we use mice expressing full-length, kinase-inactive MAP3K4 from the endogenous Map3k4 locus to examine the requirement of MAP3K4 kinase activity in sex determination. Although homozygous kinase-inactivation of MAP3K4 (Map3k4KI/KI) is lethal, a small fraction survive to adulthood. We show Map3k4KI/KI adults exhibit a 4:1 female-biased sex ratio. Many adult Map3k4KI/KI phenotypic females have a Y chromosome. XY Map3k4KI/KI adults with sex reversal display female mating behavior, but do not give rise to offspring. Reproductive organs are overtly female, but there is a broad spectrum of ovarian phenotypes, including ovarian absence, primitive ovaries, reduced ovarian size, and ovaries having follicles in all stages of development. Further, XY Map3k4KI/KI adults are smaller than either male or female Map3k4WT/WT mice. Examination of the critical stage of gonadal sex determination at E11.5 shows that loss of MAP3K4 kinase activity results in the loss of Sry expression in XY Map3k4KI/KI embryos, indicating embryonic male gonadal sex reversal. Together, these findings demonstrate the essential role for kinase activity of MAP3K4 in male gonadal sex determination. Summary sentence Male gonadal sex determination requires the kinase activity of the multi-domain containing protein MAP3K4. Graphical Abstract

We hypothesized that sexually dimorphic differences exist in the expression of miRNAs in amniotic fluid (AF) and maternal blood plasma (MP) in association with the process of sex determination and gonad differentiation in cattle. Amniotic fluid and MP were collected from six pregnant heifers (three carrying a single male and three a single female embryo) following slaughter on Day 39 postinsemination, coinciding with the peak of SRY expression. Samples (six AF and six MP) were profiled using an miRNA Serum/Plasma Focus PCR Panel. Differentially expressed (DE) miRNAs were identified in AF (n = 5) and associated MP (n = 56) of male vs. female embryos (P, Grant Support. Supported by Science Foundation Ireland (13/IA/1983) and grant RTI2018–093548-BI00 from the Spanish Ministry of Science and Innovation. IG-R was supported by a predoctoral fellowship from the Spanish Ministry of Science and Innovation (BES-2016-077794), and BP was supported by a Marie Sklodowska-Curie ITN European Joint Doctorate in Biology and Technology of Reproductive Health (REP-BIOTECH 675526).

BACKGROUND Co-morbidity of SRY gene turner syndrome (TS) with positive SRY gene and non-classical congenital adrenal hyperplasia (NCAH) is extremely rare and has never been reported to date. CASE SUMMARY In this article, we present a 14-year-old girl who was referred to our hospital with short stature (weight of 43 kg and height of 143 cm, < -2 SD) with no secondary sexual characteristics (labia minora dysplasia). Laboratory tests indicated hypergonadotropic hypogonadism with significantly increased androstenedione and 17-hydroxyprogesterone (17-OHP) levels. This was accompanied by the thickening of the extremity of the left adrenal medial limb. The patient’s karyotype was 45,X/46,X, +mar, and cytogenetic analysis using multiplex ligation-dependent probe amplification and high-throughput sequencing indicated that the SRY gene was positive with compound heterozygous mutations in CYP21A2 as the causative gene for congenital adrenal hyperplasia. The sites of the suspected candidate mutations were amplified and verified using Sanger sequencing. The patient was finally diagnosed as having SRY positive TS with NCAH. The patient and her family initially refused medical treatment. At her most recent follow-up visit (age = 15 years old), the patient presented facial hair, height increase to 148 cm, and weight of 52 kg, while androstenedione and 17-OHP levels remained high. The patient was finally willing to take small doses of hydrocortisone (10 mg/d). CONCLUSION In conclusion, upon evaluation of the patient mentioned in the report, we feel that 17-OHP measurement and cytogenetic analysis are necessary for TS patients even in the absence of significant virilization signs. This will play a significant role in guiding diagnosis and treatment.

lt;bgt;Background and Objective:lt;/bgt; The sex pre-selection for offspring before conception is desirable demand especially for the breeding program of farm animals. This study aimed to evaluate the preconception treatment of monovalent and divalent ions on the primary sex ratio, ovarian structures and serum minerals levels in New Zealand white rabbit does.lt;bgt;Materials and Methods:lt;/bgt; Nine New Zealand white rabbits doelt;igt;lt;/igt;(5.4±0.61 months of age and 2.4±0.35 kg of body weight) were used. Rabbits in the 1lt;supgt;stlt;/supgt; group were given drinking water only (control). While the 2lt;supgt;ndlt;/supgt; and 3lt;supgt;rdlt;/supgt; groups were given 1% of (calcium and magnesium) and (sodium and potassium) in daily drinking water, respectively for 15 days before mating. The embryos of each group were individually collected after three days of mating for primary sex detection using SRY (Sex Determining Region Y) Polymerase Chain Reaction (PCR) assay. Mineral analyses for all studied animals were weekly detected in serum before and after mating.lt;bgt;Results:lt;/bgt; The primary sex ratio for embryos of rabbits does receive (Na+K) produced more males (69.7%) while (Ca+Mg) administrated rabbits does produce more females (72.2%). The mineral treatment leads to a significant increase in the number of corpus luteum, total embryos, follicles bleeding and a significant decline in the count of large follicles. Also, there was no significant change in serum Na and Ca levels in the treated groups compared to the control.lt;bgt;Conclusion:lt;/bgt; The preconception administration of Ca+Mg could produce more females while Na+K could produce more males without adverse side effects on serum minerals concentration.

Objective During the prenatal period, steroidogenic factor 1 is required for the development of the adrenal glands and for gonadal determination and differentiation, and after birth, it regulates gonadal progenitor cell formation and their survival. Here, we describe the clinical phenotype of three 46,XY patients (2 brothers and an unrelated subject) with disorder of sex development due to the same genetic variant. Methods All patients underwent hormonal and pelvic ultrasound studies. Sequence analysis and deletion/duplication testing of a panel encompassing 8 genes (AR, DHH, MAP3K1, NROB1, SRD5A2, SRY, WT1, and nuclear receptor subfamily 5, group A, member 1 [NR5A1]) were performed in the index cases. All family members were tested for the presence of the NR5A1 variant. Results A variant previously described as likely pathogenic in NR5A1 (c.251G>A, p.Arg84His) that segregated in 1 family with different degrees of under-virilization was found. The family 1 index case (IV2) and his brother (IV3) had an external masculinization scale score of 5/12, but only the index case had Mullerian remnants; however, the family 2 patient had a milder score of 9/12. The older female relatives of family 1 who harbor this variant experienced premature menopause. Conclusion To our knowledge, this is the first report where the c.251G>A (p.Arg84His) variant is associated with the presence of Mullerian remnants in 46,XY subjects and primary ovarian insufficiency in 46,XX individuals. The segregation of this variant with clinical manifestations provides further evidence for considering it as pathogenic.

The clinical case of a rare variant of disorder of sex development (DSD) is described. This disorder was diagnosed with an emergency operation for the traumatic rupture of the gonad. A patient (14 years old) with a male phenotype and lack of muller duct derivatives had a female SRY negative karyotype (46,XX) and an ovotesticular gonad structure as a result of duplication in the regulatory zone of the SOX9 gene. Ovotesticular disorders of sex development with karyotype 46,XX, as a rule, are accompanied by an ambiguous genitalia and derivatives of the muller structures. Early diagnosis of the described variant of DSD was difficult due to the development of male type genitalia. Timely identification of DSD including the presented option of DSD, is possible during routine examinations of the urologist with mandatory ultrasound examination of the scrotum and pelvis.

Objective 17 α-hydroxylase/17, 20-lyase deficiency (17-OHD) is a rare recessive hereditary disease that can be attributed to cytochrome P450 17 α-hydroxylase deficiency caused by CYP17A1 gene mutations. Methods A large cohort of 10 Chinese Han patients with 17-OHD from 2012 to 2020 were enrolled. The clinical and biochemical features were investigated, and genetic mutations of CYP17A1 were analyzed by polymerase chain reaction-Sanger sequencing. Karyotype identification and the SRY gene test were also carried out. In silico analysis was used to predict the effects of genetic mutations on the protein function. Results All patients were female. Common complaints were hypertension, hypokalemia, and primary amenorrhea. The karyotype was 46, XY, and the SRY gene was detected in 7 patients; the karyotype was XX in the remaining 3 patients. A total of 7 mutations including Y329N, Y329X, Y329Lfs∗, R96W, A82D, S380N, and A487_P489del have been identified in the CYP17A1 gene. The Y329Lfs∗ mutation was found in 9/10 (90%) of patients with a high allele frequency of 70%. In silico prediction showed that a novel variant of c.1139G>A (S380N) occurs at a conserved residue and can cause disease. Conclusion We presented a detailed description of the clinical and genetic characteristics in Chinese patients with 17-OHD and concluded that Y329Lfs∗ mutation of CYP17A1 is prevalent in the Chinese Han population. Therefore, hotspot screening by polymerase chain reaction-Sanger sequencing for exon 6 of CYP17A1 could contribute to the rapid diagnosis of 17-OHD in China. Genetic counseling based on the genetic diagnosis for at-risk relatives is advised.

SOX9, a transcription factor, is expressed in the undifferentiated XX and XY gonads. SRY induces significant upregulation of SOX9 expression in XY gonads. Loss-of-function SOX9 variants cause testicular dysgenesis in 46,XY patients, while duplication of the total gene or the upstream regulatory region results in testicular development in 46,XX patients. However, gain-of-function (GoF) SOX9 variants have not been reported previously. We report the case of a 16-year-old female patient with a 46,XX karyotype who had masculinized external genitalia and unilateral ovotestis. Next-generation sequencing-based genetic screening for disorders of sex development led to the identification of a novel SOX9 variant (p.Glu50Lys), transmitted from the phenotypically normal father. Expression analysis showed that E50K-SOX9 enhanced transactivation of the luciferase reporter containing the testis enhancer sequence core element compared with that containing the wildtype-SOX9. This GoF activity was not observed in the luciferase reporter containing Amh, the gene for anti-Mullerian hormone. We genetically engineered female mice (Sox9E50K/E50K ), and they showed no abnormalities in the external genitalia or ovaries. In conclusion, a novel SOX9 variant with a promoter-specific GoF activity was identified in vitro; however, the disease phenotype was not recapitulated by the mouse model. At present, the association between the GoF SOX9 variant and the ovotestis phenotype remains unclear. Future studies are needed to verify the possible association.

Separation of X and Y chromosome-bearing sperm is an appropriate method for the selection of desired sex of offspring to increase the profit in livestock industries. The purpose of this study was the production of a monoclonal antibody against recombinant bovine sex-determining region Y protein for separation Y sperm. The hybridoma cells from splenocytes of immunized female's balb/C mice and Sp2/0 cells were made. The binding affinity of our monoclonal antibody (mAbSRY2) was compared with mouse monoclonal SRY-15. The Western blot method indicated that mAbSRY2 successfully detected the rbSRY protein. The specificity and sensitivity of mAbSRY2 is comparable to SRY-15 commercially ones. The SRY gene in 100% of bull semen contains the Y chromosome that had the strongest binding affinity to mAbSRY2 was synthesized. In other words, the binding affinity of semen contains the X sperms near the negative control. In general, this immunological method can help to separate X from Y sperms. However, the mAbSRY2 is bind to Y-bearing sexed sperm, but in the future; the sexed sperms need to apply in farms.

Mammalian male sex differentiation is triggered during embryogenesis by the activation of the Y-linked testis-determining gene SRY. Since insufficient or delayed expression of SRY results in XY gonadal sex reversal, accurate regulation of SRY is critical for male development in XY animals. In humans, dysregulation of SRY may cause disorders of sex development. Mouse Sry is the most intensively studied mammalian model of sex determination. Sry expression is controlled in a spatially and temporally stringent manner. Several transcription factors play a key role in sex determination as trans-acting factors for Sry expression. In addition, recent studies have shown that several epigenetic modifications of Sry are involved in sex determination as cis-acting factors for Sry expression. Herein, we review the current understanding of transcription factor- and epigenetic modifier-mediated regulation of SRY/Sry expression.

Isodicentric chromosomes are a frequently appearing abnormality in the human Y chromosome. Making predictions regarding the phenotypic outcomes of a variety of duplications/deletions in the dicentric Y chromosome generally depends on the breakpoint location and also the level of mosaicism (45, X cell lines); in certain instances, these may not be detected and result in variations running between male, abnormal female, or ambiguity in individual genitalia. A referral was received from a urology clinic concerning two patients, one aged 34 and one aged 35, with a request to investigate reasons for infertility: G-banded karyotyping and fluorescence in situ hybridization (FISH) revealed the presence of an isodicentric Y chromosome.

The development of sexually dimorphic gonads is a unique process that starts with the specification of the bipotential genital ridges and culminates with the development of fully differentiated ovaries and testes in females and males, respectively. Research on sex determination has been mostly focused on the identification of sex determination genes, the majority of which encode for proteins and specifically transcription factors such as SOX9 in the testes and FOXL2 in the ovaries. Our understanding of which factors may be critical for sex determination have benefited from the study of human disorders of sex development (DSD) and animal models, such as the mouse and the goat, as these often replicate the same phenotypes observed in humans when mutations or chromosomic rearrangements arise in protein-coding genes. Despite the advances made so far in explaining the role of key factors such as SRY, SOX9, and FOXL2 and the genes they control, what may regulate these factors upstream is not entirely understood, often resulting in the inability to correctly diagnose DSD patients. The role of non-coding DNA, which represents 98% of the human genome, in sex determination has only recently begun to be fully appreciated. In this review, we summarize the current knowledge on the long-range regulation of 2 important sex determination genes, SOX9 and FOXL2, and discuss the challenges that lie ahead and the many avenues of research yet to be explored in the sex determination field.

As a common malignant tumor in neurosurgery, glioma is characterized as high incidence rate, easy to invade, metastasize and recurrent. It is difficult to treat and has a poor prognosis. The gliomas pathogenesis is complex and has not been fully resolved. Therefore, finding effective molecular targets for glioma is beneficial to improve therapeutic effect. The SRY-related high mobility group box 9 (SOX9) gene involves in mammalian development and is significantly increased in glioma. However, SOX9’s role in gliomas is unclear. The glioma cell line U87 was assigned into control group, scramble group that was transfected with siRNA negative control, and SOX9 siRNA group that was transfected with SOX9 siRNA followed by analysis of SOX9 mRNA and protein level by qPCR and Western blot, cell proliferation by MTT assay, cell apoptosis by Caspase 3 activity assay, cell invasion by Transwell assay, and MMP-9 level by ELISA. SOX9 siRNA transfection significantly downregulated SOX9 mRNA and protein expressions, inhibited U87 cell proliferation, enhanced Caspase 3 activity, suppressed cell invasion of U87, decreased the secretion of MMP-9 in the supernatant, and reduced ERK1/2 and P38 phosphorylation levels (P < 0.05). SOX9 can regulate the progression of glioma by regulating ERK/P38 signaling pathway, promoting cell apoptosis, inhibiting cell proliferation, and restraining cell invasion.

At embryonic day (E) 10.5, prior to gonadal sex determination, XX and XY gonads are bipotential and able to differentiate into either a testis or an ovary. At this point, they are transcriptionally and morphologically indistinguishable. Sex determination begins around E11.5 in the mouse when the supporting cell lineage commits to either Sertoli or granulosa cell fate. Testis-specific factors such as SRY and SOX9 drive differentiation of bipotential-supporting cells into the Sertoli cell pathway, whereas ovary-specific factors like WNT4 and FOXL2 guide differentiation into granulosa cells. It is known that these 2 pathways are mutually antagonistic, and repression of the alternative fate is critical for maintenance of the testis or ovary programs. While we understand much about the transcription factor networks guiding the process of sex determination, it is only more recently that we have begun to understand how this process is epigenetically controlled. Studies in the past decade have demonstrated the importance of the chromatin state for gene expression and cell fate commitment, with histone modifications and DNA accessibility having a direct role in gene regulation. It is now clear that the chromatin state during sex determination is dynamic and likely critical for the establishment and/or maintenance of the transcriptional programs. Prior to sex determination, supporting cells have similar chromatin structure and histone modification profiles, reflecting the bipotential nature of these cells. After differentiation to Sertoli or granulosa cells, the chromatin state acquires sex-specific profiles. The proteins that regulate the deposition of histone modifications or the opening of compact chromatin likely play an important role in Sertoli and granulosa cell fate commitment and gonad development. Here, we describe studies profiling the chromatin state during gonadal sex determination and one example in which depletion of Cbx2, a member of the Polycomb Repressive Complex 1 (PRC1), causes male-to-female sex reversal due to a failure to repress the ovarian pathway.

Introduction: Ovotesticular disorder of sex development is a rare condition by the concomitant presence of testicular and ovarian tissue, and usually presents genital ambiguity. Are chromosomally heterogeneous, and cytogenetic analyses is relevant. Objective: report on a patient from Manaus, Amazonas state with ovotesticular disorder of sex differentiation 46, XX and SRY-negative. Case report: Patient of 19 years, first child of non-consanguineous parents. At birth, the patient was diagnosed with genital ambiguity and, without early diagnosis, he was registered as being of the male sex. The patient underwent surgery to correct bilateral cryptorchidism, orchiopexy and colpectomy. During puberty, he developed female and male sexual characteristics. Endocrinological (normal total testosterone and estradiol as high follicle-stimulating hormone and luteinizing hormone), histopathological (right gonad, ovarian follicles and left gonads, atrophic testicles), karyotype (46, XX) and molecular (SRY-negative). Diagnosis of ovotesticular disorder of sex development was established. The patient chose to remain male and underwent bilateral mastectomy, vaginal colpectomy and bilateral gonadectomy. Currently, the patient receives hormonal replacement therapy, follow-up with a multi-professional approach and awaits masculinizing genitoplasty. Discussion: In diagnostic research, cytogenetic and molecular analysis are primary tools. For OT-DSD individuals with 46, XX, the female sex is suggested as the best sex option. Unlike the reported cases, the patient chose the male sex, since the sex at registration of birth was important in his choice. Conclusion: Cytogenetic and molecular analyses allowed us to assist in the etiological diagnosis of the patient with OT-DSD from Manaus. However, molecular analyses are necessary to elucidate the genes involved in the sexual determination of this patient.

BACKGROUND 45,X/46,XY mosaicism is a rare chromosomal abnormality with a wide range of phenotypes in both males and females, from normal individuals with different degrees of genital ambiguity to those who show signs of Turner’s syndrome. More rarely, cases of 45,X/46,XY mosaicism with a normal-appearing male phenotype are not found until a chromosome test is performed to investigate the cause of male infertility. CASE SUMMARY In this study, a 29-year-old male patient with complete azoospermia is reported. Chromosomal analyses of his lymphocytes revealed the karyotype 45,X[93%]/46,X,+mar(Y)[7%]. In addition, Y chromosome-specific markers, such as SRY, ZFY, AZFa, AZFb and AZFc, were not observed in his blood DNA according to multiplex polymerase chain reaction test. A literature review identified several 45,X/46,XY cases with a normal-appearing male phenotype, most of whom were diagnosed during infertility investigation. However, the present case is the first SRY-negative 45,X/46,XY male case diagnosed during a premarital medical examination. CONCLUSION This finding further suggests that sex determination is a complex process regulated by multiple genetic and environmental factors.

Background: The sex determination process requires distinct signaling pathways to generate either testis or ovaries from the same precursor structures, the primordial gonad. Deviations of this signaling mechanism may result in disorders/differences of sex development (DSD). The 46, XX testicular DSD is a rare genetic condition identified by a discrepancy between genetic and phenotypic sex caused sex reversal syndrome. Case Presentation: We describe the case of a 5 years-old 46, XX boy with ambiguous genitalia. On physical examination he had severe hypospadias, bifid scrotum, micropenis and palpable bilateral testes. Cytogenetic analysis of patient reveals a 46, XX karyotype. Hormonal assay showed low level of FSH, LH and Testosterone and there was no evidence of Mullerian structures based on pelvic imaging. The histopathology of gonadal tissue showed a Leydig cell hyperplasia which gives the impression of Sertoli cell nodule. Polymerase chain reaction (PCR) analysis failed to identify the presence of SRY gene, therefore a diagnosis of 46, XX Testicular DSD with SRY-negative was established. Conclusion: This report presents a rare case of SRY-negative 46, XX Testicular DSD in a boy with ambiguous genitalia. A comprehensive management including clinical, cytogenetic and molecular analyses have indicated that undiscovered genetic or environmental factors needs to be elucidated. It is important to carry out further molecular testing to establish precise diagnosis of DSD and to provide appropriate genetic counseling for patients and their family.

The sex-determining region Y (SRY) gene is a key gene involved in male sex differentiation and development. Patients with 46,XY disorders of sex development related to mutations in the high mobility group (HMG) box typically present with complete gonadal dysgenesis. In this study, we report a case of novel missense mutation c.T281G within the HMG domain of SRY in a 15-year-old patient of the female gender with 46,XY partial gonadal dysgenesis (PGD). The novel missense mutation caused the substitution of codon 94 for leucine in the HMG box of the SRY protein with an arginine codon. Leucine and arginine are aliphatic amino acids, and three-dimensional protein structure prediction revealed only slight structural changes in the SRY protein. Thus, the SRY protein had maintained some of its functions, and the patient presented with PGD. In conclusion, we identified a novel SRY mutation in a patient with 46,XY PGD. Based on the protein model, we believe that the mutation in the HMG domain helped to maintain the partial function of the SRY protein. The condition of our patient differed from the well-known 46,XY complete gonadal dysgenesis caused by mutations in the HMG region. In fact, this is the first case of 46,XY PGD caused by mutations in the HMG region to be reported, and therefore, our experience has expanded the mutation spectrum of the SRY gene. Furthermore, the present case demonstrates that mutations located in the HMG domain of SRY gene cannot be ruled out in patients with a clinical diagnosis of 46,XY PGD.

Background Turner syndrome (TS) has a variety of different karyotypes, with a wide range of phenotypic features, but the specific karyotype may not always predict the phenotype. TS with Y chromosome mosaicism may have mixed gonadal dysgenesis, and the mosaicism is related to the potential for gonadoblastoma. Case summary In this case report, we report two cases of TS with different karyotypes and gonadal dysgenesis. Patient 1 had obvious virilization, and was positive for the SRY gene, but her karyotype in peripheral blood lymphocytes was 45X. Patient 2 had a mosaic karyotype, 45X/46X, dic (Y:Y) (p11.3:p11.2), and the proportion of Y-bearing cells was 50% in peripheral blood lymphocytes, but the patient had normal female external genitalia and streaky gonads, with no genital virilism. Different tissues in the same TS individual may exhibit different ratios of mosaicism. The gonadal determination and differentiation of mosaic TS are primarily dependent on the predominant cell line in the gonads. Conclusion In TS patients with virilization, it is necessary to test at least two to three tissues to search for cryptic Y material.

Background Sex determination can be useful in forensic casework, such as in mass disasters, transportation accidents, and cases of a missing person or sexual assault. The remnants of the body can be traced by DNA of the victim, using samples from various sources such as teeth, oral epithelial tissue, and saliva. Main body The review aimed to describe research in forensic dentistry with DNA source from the oral region and methods of the applied DNA analysis. A search in PubMed, Google Scholar, and Scopus electronic databases from 2009 to 2019 was conducted to include studies according to PRISMA guidelines. Ten studies were eligible for the review. Genetic markers originated from dentin, dental pulp, saliva, or epithelial cells from buccal tissue and prosthesis. The applied DNA analysis methods were PCR, real-time PCR, and nested PCR. Conclusions The published articles mostly showed successful DNA extraction and sex determination, but the rate of success declined as the sample source underwent manipulation to mimic the forensic conditions. Amelogenin, SRY, and DYS14 were reliable indicators for sex determination. Molecular analysis has proved to be efficient and accurate, but the daily forensic practice must select the most appropriate method according to the available body remnants.

Two rather than one For several decades, it has been believed that the mammalian sex-determining gene Sry contains a single exon. Miyawaki et al. have now identified a cryptic second exon of mouse Sry . Loss- and gain-of-function analyses revealed that the two-exon SRY (SRY-T), not the canonical single exon–encoded SRY (SRY-S), is the bona fide testis-determining factor. Sry exon2 is composed of retrotransposon-derived sequences. The SRY-S carboxyl terminus contains a degradation sequence (degron), whereas the SRY-T carboxyl terminus encoded in the Sry exon2 is degron free, thereby conferring protein stability on SRY-T. Science , this issue p. 121

Background Noninvasive fetal sex determination by analyzing Y chromosome-specific sequences is very useful in the management of cases related to sex-linked genetic diseases. The aim of this study was to establish a non-invasive fetal sex determination test using Real-Time PCR and specific probes. Methods The study was a prospective observational cohort study conducted from August 2018 to September 2019. Venous blood samples were collected from 25 Iranian pregnant women at weeks 7 to 25 of gestation. Cell-free DNA (cfDNA) was isolated from the plasma of samples and fetal sex was determined by SRY gene analysis using the Real-Time PCR technique. In the absence of SRY detection, the presence of fetal DNA was investigated using cfDNA treated with BstUI enzyme and PCR for the epigenetic marker RASSF1A. Results Of the total samples analyzed, 48% were male and 52% female. The RASSF1A assay performed on SRY negative cases also confirmed the presence of cell-free fetal DNA. Genotype results were in full agreement with neonate gender, and the accuracy of noninvasive fetal sex determination was 100%. Conclusion Fetal sex determination using the strategy applied in this study is noninvasive and highly accurate and can be exploited in the management of sex-linked genetic diseases.

In contrast to established zebrafish gene annotations, the question of sex determination has still not been conclusively clarified for developing zebrafish, Danio rerio, larvae, 28 dpf or earlier. Recent studies indicate polygenic sex determination (PSD), with the genes being distributed throughout the genome. Early genetic markers of sex in zebrafish help unravel co-founding sex-related differences to apply to human health and environmental toxicity studies. A qPCR-based method was developed for six genes: cytochrome P450, family 17, subfamily A, polypeptide 1 (cyp17a1); cytochrome P450, family 19, subfamily A, polypeptide 1a (cyp19a1a); cytochrome P450, family 19, subfamily A, polypeptides 1b (cyp19a1b); vitellogenin 1 (vtg1); nuclear receptor subfamily 0, group B, member 1 (nr0b1), sry (sex-determining region Y)-box 9b (sox9b) and actin, beta 1 (actb1), the reference gene. Sry-box 9a (Sox9a), insulin-like growth factor 3 (igf3) and double sex and mab-3 related transcription factor 1 (dmrt1), which are also known to be associated with sex determination, were used in gene expression tests. Additionally, Next-Generation-Sequencing (NGS) sequenced the genome of two adult female and male and two juveniles. PCR analysis of adult zebrafish revealed sex-specific expression of cyp17a1, cyp19a1a, vtg1, igf3 and dmrt1, the first four strongly expressed in female zebrafish and the last one highly expressed in male conspecifics. From NGS, nine female and four male-fated genes were selected as novel for assessing zebrafish sex, 28 dpf. Differences in transcriptomes allowed allocation of sex-specific genes also expressed in juvenile zebrafish. Electronic supplementary material The online version of this article (10.1007/s00204-020-02915-y) contains supplementary material, which is available to authorized users.

The detection of fetal cell-free DNA (cfDNA) from maternal plasma has enabled the development of essential techniques in prenatal diagnosis during recent years. Extracellular vesicles including exosomes were determined to carry fetal DNA fragments. Considering the known difficulties during isolation and stability of cfDNA, exosomes might provide a new opportunity for prenatal diagnosis and screening. In this study, comparison of cfDNA and exosome DNA (exoDNA) for predicting the fetal sex and Rhesus D (RHD) genotype was performed by using real-time polymerase chain reaction with simultaneous amplification of sequences of SRY and RHD genes. Fetal sex and RHD were determined in 100 and 81 RHD-negative pregnant women with cfDNA and exoDNA, respectively. The gestation ages of pregnant women were between 9 and 40 weeks. The results were compared with the neonatal phenotype for gender and a serological test for RHD. The cfDNA revealed 95.75% sensitivity and 100% specificity in RHD positivity and 100% sensitivity and 95.45% specificity in SRY positivity. Cohen’s agreement coefficient in the Kappa test ranged from 0.8 to 1.0 (P

AIMS Confirmation of a breast origin for triple-negative breast cancer (TNBC) is sometimes problematic. The traditional breast markers GATA-binding protein 3 (GATA3), mammaglobin (MGB) and gross cystic disease fluid protein 15 (GCDFP15) have shown limitations in identifying TNBC. Here, we aimed to examine the diagnostic potential of the newly proposed TNBC marker, Sry-related high-mobility-group/HMG box 10 (SOX10). METHODS AND RESULTS We analysed and compared SOX10 expression with GATA3, MGB and GCDFP15 expression in a test cohort of 1838 invasive breast cancers (IBCs) by using tissue microarrays. The findings from the test cohort were further examined with a validation cohort of 42 TNBCs in whole sections. The overall expression rates of SOX10, GATA3, MGB and GCDFP15 were 6.9%, 83.1%, 47.0%, and 34.8%, respectively. Among the TNBCs within this cohort, the expression rates of SOX10, GATA3, MGB and GCDFP15 were 31.3%, 34.5%, 27.9%, and 25.2%, respectively. SOX10 was strongly associated with TNBC (P

Fibroblast growth factor 9 (FGF9) is an autocrine/paracrine growth factor that plays critical roles in embryonic and organ developments and is involved in diverse physiological events. Loss of function of FGF9 exhibits male-to-female sex reversal in the transgenic mouse model and gain of FGF9 copy number was found in human 46, XX sex reversal patient with disorders of sex development. These results suggested that FGF9 plays a vital role in male sex development. Nevertheless, how FGF9/Fgf9 expression is regulated during testis determination remains unclear. In this study, we demonstrated that human and mouse SRY bind to −833 to −821 of human FGF9 and −1010 to −998 of mouse Fgf9, respectively, and control FGF9/Fgf9 mRNA expression. Interestingly, we showed that mouse SRY cooperates with SF1 to regulate Fgf9 expression, whereas human SRY-mediated FGF9 expression is SF1 independent. Furthermore, using an ex vivo gonadal culture system, we showed that FGF9 expression is sufficient to switch cell fate from female to male sex development in 12–16 tail somite XX mouse gonads. Taken together, our findings provide evidence to support the SRY-dependent, fate-determining role of FGF9 in male sex development.

The 46,XX ovotesticular disorder of sex development (DSD) is rarely observed in humans. This disorder is generally described as ambiguous genitalia with the presence of ovarian and testicular tissues in different gonads or in the same gonad. Almost no subjects with 46,XX ovotesticular DSD have sex-determining region of the Y chromosome (SRY) gene. It is known that excessive expression of SRY-related high mobility group box 9 (SOX9) is the cause of SRY-negative 46,XX ovotesticular DSD in the absence of SRY. Here, we analyzed our SRY-negative case with 46,XX ovotesticular DSD. In an array comparative genomic hybridization study using a peripheral blood sample from the patient, a duplication of 1114 kb (Hg19 coordinates: chr17:69006280-70120619) in the region of 17q24.3 containing SOX9 was detected. This is the first case reported from Turkey, exhibiting SOX9 duplication in SRY-negative 46,XX ovotesticular DSD.

Male-to-female sex reversal in horses is a developmental disorder in which phenotypic females have a male genetic constitution. Male-to-female sex reversal is the second most common genetic sex abnormality, after X chromosome monosomy. All male-to-female sex reversal cases studied to date have been found to be infertile. Therefore, a screening test is particularly useful in laboratories doing DNA genotyping in horses. Our laboratory has tested > 209,000 horses for parentage using a panel of microsatellite markers and the sex marker gene amelogenin ( AMEL). Suspect XY sex reversal cases are reported females with a male profile by AMEL testing. After routine genotyping, 49 cases were detected and further tested using the sex-determining region Y ( SRY) gene, confirming the XY SRY-negative genotype of suspect sex reversal cases. When some inconsistencies arose in the initial result, a molecular panel of X- and Y-linked markers was analyzed for these samples. Of the 49 cases, 33 were confirmed as XY SRY-negative. The remaining 16 cases were identified as false-positives as a result of anomalies of AMEL testing in horses.

Sex is an important biological variable as many physiological as well as disease processes differ between females and males. The fundamental biological distinction between females and males starts with chromosomal sex, and the establishment of XX and XY cells and tissues. Polymerase Chain Reaction (PCR) is a simple and effective method to easily determine chromosomal or genetic sex of cells and tissues. The goal of this study was to develop a simple multiplex PCR genotyping assay to distinguish XX and XY tissues in sheep. Primers were designed to amplify a fragment of the autosomal gene myogenin (MYOG) and sex determining region on the Y chromosome (SRY). PCR analysis was performed on a variety of genomic DNA samples isolated from fetal sheep skeletal muscle, brain, liver, and placenta, and revealed a single 259 bp band for MYOG in XX females, and a 259 bp band for MYOG and a 167 bp band for SRY in XY males. Amplicons were clearly distinguishable by gel electrophoresis, and their sequences revealed 100% identity to the known ovine MYOG and SRY sequence. The reported multiplex PCR genotyping assay provides a rapid means to distinguish XX and XY sheep tissues using low volume samples.