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Noninvasive sampling can provide an efficient means of genetically monitoring mammals. Due to the fragmented quality of DNA derived from such samples, few methods span taxonomic groups beyond species. Here, we describe a more universal protocol for the molecular sex identification of order Carnivora. PCR amplification of a 176 bp segment of the SRY gene indicates a male, whereas no amplification indicates a female. A 245 bp segment of the ZFX gene serves as a positive control. This method is internally consistent, and multiple trials confer confidence in results. It applies to at least three families (Canidae, Felidae, and Phocidae) in both carnivore suborders (Feliformia and Caniformia), rendering this protocol the first sex identification method with amplicon sizes appropriate for noninvasive samples suitable for multiple families within order Carnivora. [ABSTRACT FROM AUTHOR]

Patients with disorders of sex development (DSD), especially those with gonadal dysgenesis and hypovirilization, are at risk of developing the so-called type II germ cell tumors (GCTs). Both carcinoma in situ and gonadoblastoma (GB) can be the precursor lesion, resulting in a seminomatous or non-seminomatous invasive cancer. SRY mutations residing in the HMG domain are found in 10–15% of 46,XY gonadal dysgenesis cases. This domain contains two nuclear localization signals (NLSs). In this study, we report a unique case of a phenotypical normal woman, diagnosed as a patient with 46,XY gonadal dysgenesis, with an NLS missense mutation, on the basis of the histological diagnosis of a unilateral GB. The normal role of SRY in gonadal development is the upregulation of SOX9 expression. The premalignant lesion of the initially removed gonad was positive for OCT3/4, TSPY and stem cell factor in germ cells, and for FOXL2 in the stromal component (ie, granulosa cells), but not for SOX9. On the basis of these findings, prophylactical gonadectomy of the other gonad was performed, also showing a GB lesion positive for both FOXL2 (ovary) and SOX9 (testis). The identified W70L mutation in the SRY gene resulted in a 50% reduction in the nuclear accumulation of the mutant protein compared with wild type. This likely explains the diminished SOX9 expression, and therefore the lack of proper Sertoli cell differentiation during development. This case shows the value of the proper diagnosis of human GCTs in identification of patients with DSD, which allows subsequent early diagnosis and prevention of the development of an invasive cancer, likely to be treated by chemotherapy at young age. [ABSTRACT FROM AUTHOR]

Proteomic analysis of developmental toxicity by two-dimensional electrophoresis (2-DE) may detect gender-related toxic effects in embryos without visible gender characteristics. In the present study, we explored sexing of rat embryo stored in frozen 2-DE samples by polymerase chain reaction (PCR) of a male-specific gene sequence, sex determining region Y (Sry). The embryo proper and yolk sac membrane at gestation day 11 from Wistar rats were used for stored embryonic 2-DE samples. The embryonic 2-DE samples were desalted and their total DNA was extracted. The Sry sequence in the extracted DNA was amplified by PCR and the product was analyzed by agarose gel electrophoresis. The embryos with the PCR product of Sry were determined as male, and those without the product were determined as female. It was concluded that stored embryonic 2-DE samples could be used for retrospective examination of gender-related effects in proteomic analysis of developmental toxicity. [ABSTRACT FROM AUTHOR]

Individuals with male phenotypes and 46 XX karyotype appear in about 1 of 20,000 births with clinical features varying from normal male appearance to sexual ambiguity and hermaphroditism. More than 80% of these patients present a spontaneous translocation of the SRY gene from the Y to the X chromosome in the paternal germinal cells. We present a case of a 2 years old boy diagnosed with minor hypospadias, bifid scrotum, normal penis and palpable gonads in the scrotum. The karyotype is 46 XX and FISH analysis reveals SRY translocation on one of the X chromosomes. Ultrasound exam does not reveal any mullerian structures and a hCG test proves the testes to be functional. A short course of treatment with hCG is recommended in order to induce the proper development of the scotal sac. The patient will need monitoring, in order to identify the development of hypergonadic hypogonadism, which characterizes such patients in later life. This case underlines the importance of comprehensively investigating any patient with even minor genitalia anomalies. [ABSTRACT FROM AUTHOR]

Abstract: A 4-year-old Icelandic horse, considered to be a mare, showed stallion-like behavior in a group of mares. On clinical examination, the horse turned out to have an enlarged erectable phallic clitoris. Ultrasound examination showed a normal-sized left ovary covered with numerous small follicular cysts and a compact testis-like tissue in place of the right ovary. The karyotype was normal for a mare (64,XX), and the horse was found to be negative for the Y chromosome–specific markers SRY, ZFY, and EIF1AY. This case indicates that the intersexual phenotype may be caused by autosomal recessive mutation, resulting in defects in cortisol biosynthesis rather than transferal of Y chromosome male–specific genes. This is the first report of an intersexual phenotype in an Icelandic horse that is likely to be a true hermaphrodite because of female sex chromosomes and a mixture of female and male gonads and external genitals. [Copyright &y& Elsevier]

Abstract: The SRY gene (sex-determining region on the Y chromosome) was isolated in 1990 and is known as the testis-determining factor on the Y chromosome. The SRY has been considered as a transcription factor since it contains an HMG box, which functions as a DNA-binding domain. However, a direct target for SRY remains to be identified. We have investigated the function of SRY through proteomics and transcriptome approaches, and by using two stable SRY-overexpressing cell lines (SRY1 and SRY2) in NT2/D1 cells derived from human testicular embryonal cell carcinoma. The results of 2-dimensional gel electrophoresis show that SRY overexpression causes a considerable downregulation of many chaperone proteins. SRY also upregulates laminin, which is important for Sertoli cell differentiation. Additionally, transcriptome analysis shows that SRY overexpression upregulates many zinc finger proteins and downregulates cellular growth factors with S or G2/M arrest of the cell cycle and inhibition of cellular proliferation. [Copyright &y& Elsevier]

Sexing wild marine mammals that show little to no sexual dimorphism is challenging. For sirenians that are difficult to catch or approach closely, molecular sexing from tissue biopsies offers an alternative method to visual discrimination. This paper reports the results of a field study to validate the use of two sexing methods: (1) visual discrimination of sex vs (2) molecular sexing based on a multiplex PCR assay which amplifies the male-specific SRY gene and differentiates ZFX and ZFY gametologues. Skin samples from 628 dugongs (Dugong dugon) and 100 Florida manatees (Trichechus manatus latirostris) were analysed and assigned as male or female based on molecular sex. These individuals were also assigned a sex based on either direct observation of the genitalia and/or the association of the individual with a calf. Individuals of both species showed 93 to 96% congruence between visual and molecular sexing. For the remaining 4 to 7%, the discrepancies could be explained by human error. To mitigate this error rate, we recommend using both of these robust techniques, with routine inclusion of sex primers into microsatellite panels employed for identity, along with trained field observers and stringent sample handling. [ABSTRACT FROM AUTHOR]

Because of their exceptionally long independent evolution, a range diminution of their Eocene relatives, and a remarkable subsequent diversification in Southeast Asia, tarsiers are of particular importance to evolutionary primatologists. Little is known, however, on the processes shaping the radiation of these small enigmatic primates--especially on the Indonesian island of Sulawesi, their center of endemism. Geological reconstructions and progress in applying DNA sequence information to divergence dating now provide us with the tools and background to comprehend tarsier dispersal. Here, we describe effects of plate-tectonic movements, Pleistocene sea level changes, and hybridization on the divergence of central Sulawesi tarsiers. We analyzed 12 microsatellites, the cytochrome b gene, the hypervariable region I of the mitochondrial control region, and the sex-determining region on the Y-chromosome from 144 specimens captured along a transect crossing a species boundary and a contact zone between 2 microplates. Based on these differentially inherited genetic markers, geographic information, and recordings of vocalizations, we demonstrate that the species boundary coincides with a tectonic suture. We estimate the most recent common ancestor of the 2 taxa to have lived 1.4 Mya, we describe asymmetrical introgressive hybridization, and we give evidence of unbiased dispersal in one species and male-biased dispersal in another species. This study exemplifies that the distribution of tarsier acoustic forms on Sulawesi is consistent with the allocation of genetic variability and that plate-tectonic and glacial events have left traceable marks in the biogeography of this island's unique fauna. [ABSTRACT FROM AUTHOR]

Abstract: Ovotestis development in B6-XYPOS mice provides a rare opportunity to study the interaction of the testis- and ovary-determining pathways in the same tissue. We studied expression of several markers of mouse fetal testis (SRY, SOX9) or ovary (FOXL2, Rspo1) development in B6-XYPOS ovotestes by immunofluorescence, using normal testes and ovaries as controls. In ovotestes, SOX9 was expressed only in the central region where SRY is expressed earliest, resulting in testis cord formation. Surprisingly, FOXL2-expressing cells also were found in this region, but individual cells expressed either FOXL2 or SOX9, not both. At the poles, even though SOX9 was not up-regulated, SRY expression was down-regulated normally as in XY testes, and FOXL2 was expressed from an early stage, demonstrating ovarian differentiation in these areas. Our data (1) show that SRY must act within a specific developmental window to activate Sox9; (2) challenge the established view that SOX9 is responsible for down-regulating Sry expression; (3) disprove the concept that testicular and ovarian cells occupy discrete domains in ovotestes; and (4) suggest that FOXL2 is actively suppressed in Sertoli cell precursors by the action of SOX9. Together these findings provide important new insights into the molecular regulation of testis and ovary development. [Copyright &y& Elsevier]

Abstract: Mutations of SRY are the cause of 46,XY complete pure gonadal dysgenesis (PGD) in 10–15% of patients. In this study, DNA was isolated and sequenced from blood leukocytes and from paraffin-embedded gonadal tissue in five patients with 46,XY complete PGD. DNA binding capability was analyzed by three different methods. The structure of the full length SRY and its mutant proteins was carried out using a protein molecular model. DNA analysis revealed two mutations and one synonymous polymorphism: in patient #4 a Y96C mutation, and a E156 polymorphism; in patient #5 a S143G mosaic mutation limited to gonadal tissue. We demonstrated, by all methods used, that both mutant proteins reduced SRY DNA binding activity. The three-dimensional structure of SRY suggested that besides the HMG box, the carboxy-terminal region of SRY interacts with DNA. In conclusion, we identified two SRY mutations and a polymorphism in two patients with 46,XY complete PGD, demonstrating the importance of the carboxy-terminal region of SRY in DNA binding activity. [Copyright &y& Elsevier]

Abstract: The ability of adult peripheral sensory neurons to undergo functional and anatomical recovery following nerve injury is due in part to successful activation of transcriptional regulatory pathways. Previous in vitro evidence had suggested that the transcription factor Sox11, a HMG-domain containing protein that is highly expressed in developing sensory neurons, is an important component of this regenerative transcriptional control program. To further test the role of Sox11 in an in vivo system, we developed a new approach to specifically target small interfering RNAs (siRNAs) conjugated to the membrane permeable molecule Penetratin to injured sensory afferents. Injection of Sox11 siRNAs into the mouse saphenous nerve caused a transient knockdown of Sox11 mRNA that transiently inhibited in vivo regeneration. Electron microscopic level analysis of Sox11 RNAi-injected nerves showed that regeneration of myelinated and unmyelinated axons was inhibited. Nearly all neurons in ganglia of crushed nerves that were Sox11 immunopositive showed colabeling for the stress and injury-associated activating transcription factor 3 (ATF3). In addition, treatment with Sox11 siRNAs in vitro and in vivo caused a transcriptional and translational level reduction in ATF3 expression. These anatomical and expression data support an intrinsic role for Sox11 in events that underlie successful regeneration following peripheral nerve injury. [Copyright &y& Elsevier]

Sox genes share a highly conserved DNA-binding motif, the HMG (high mobility group)-box domain, and have diverse roles in vertebrate embryonic development. A novel SRY-related cDNA (temporarily called Sox33) isolated from the Chinese alligator ( Alligator sinensis) is 1,819 bp in length, with an open reading frame from 220 to 1113 bp, encoding a protein of 298 amino acids. Two putative polyadenylation signal sequences (AATAAA) are present upstream of the poly(A) tail in the 3′ UTR (at 1255–1260 and 1774–1779). The putative protein contains an HMG-box domain most closely related to hSox12, mSox4, rtSox11, and mSox11 homologs, indicating that alligator Sox33 belongs to group C in the Sox gene family. Alligator adult and developing tissues were tested for Sox33 mRNA by independent Northern blots using a 336-bp probe (at 907–1243) between the HMG-box and the poly(A) site I and a 277-bp probe (at 1477–1754) between the two polyadenylation sites. Two transcripts (1.3 kb and 1.8 kb) in developing brain and one (1.8 kb) in adult brain were identified by the 336-bp probe; only one transcript (1.8 kb) in developing and adult brains was detected by the 277-bp probe. The results suggest that alligator Sox33 may use a different polyadenylation mechanism in the developing brain and play a role in the development and maintenance of the nervous system. [ABSTRACT FROM AUTHOR]

Sex determination varies greatly among animal species including mammals. Although two identical (XX) or distinct (XY) gonosomes are found in all species of the latter, the ratio of sex-specific content varies considerably ranging from a small minority in monotremes to more than 90% in primates. However, the similarities are strong enough to allow us to study those mechanisms in comparison with each other and obtain useful information on the evolution of sex determination. The paper focuses on the basic principles of sex determination in mammals, stressing the common evolutionary background behind the striking similarities observed even among phenotypically and geographically distinct species. Sequencing and comparative genetic data seem to suggest that a common evolutionary origin for all mammalian sex determination strategies is most probable. Nevertheless, original research data is still required for the understanding of the complex underlying mechanisms. [ABSTRACT FROM AUTHOR]

Humans, mice, and even kangaroos have an XX female:XY male system of sex determination, in which the Y harbors a male-dominant sex-determining gene SRY. Birds have the opposite, ZZ males and ZW females, and may use a dosage-sensitive Z-borne gene. Other reptiles have genetic sex but no visible sex chromosomes, or determine sex by temperature of egg incubation. How can we make sense of so much variation? How do systems change in evolution? Studies of some unlikely animals-platypus and dragon lizards, frogs and fish-confirm that evolutionary transitions have occurred between TSD and GSD systems, between XY and ZW systems, and even between male and female heterogametic systems. Here I explore nonniodel systems that offer some new perspectives on some venerable questions of sex and sex chromosomes. [ABSTRACT FROM AUTHOR]

Abstract: In most mammals, sex is determined by the presence or absence of the SRY gene. SRY encodes a DNA-binding HMG-box transcription factor which, during embryogenesis, is the initial trigger of testis differentiation from the bipotential gonad, yet its precise mode of function remains unclear. In ovarian development, R-spondin1 and Wnt4 act through the Wnt/β-catenin-signaling pathway to regulate TCF-dependent expression of unknown target genes and repress testis development. Conversely, SRY may be necessary to prevent the development of ovaries by inhibiting the action of ovarian-determining genes. We hypothesize that SRY prevents Wnt/β-catenin signaling, thereby inhibiting ovarian development. In HEK293T cells, SRY repressed β-catenin-mediated TCF-dependent gene activation in the presence of a specific GSK3β inhibitor or an activated β-catenin mutant, suggesting that SRY inhibits Wnt signaling at the level of β-catenin. Three SRY mutant proteins with nuclear localization defects, encoded by XY male-to-female patients, failed to inhibit β-catenin; surprisingly four SRY sex reversed mutants with defective DNA-binding activity showed near wild-type SRY inhibitory activity. Moreover the potent transactivator SRY–VP16 fusion protein also showed wild-type SRY inhibitory activity. Thus SRY inhibition of β-catenin involves neither DNA-binding nor transactivation functions of SRY. β-Catenin and SRY interact in vitro and SRY expression triggered β-catenin localization into specific nuclear bodies in NT2/D1 and Hela cells. We conclude that SRY inhibits β-catenin-mediated Wnt signaling by a novel nuclear function of SRY that could be important in sex determination. [Copyright &y& Elsevier]

Introduction: Isodicentric (idic) chromosomes are the most common Y structural abnormalities and their influence on gonadal and somatic development is extremely variable. The prediction of their phenotypic consequences is often difficult because of the variety of genomic sequences concerned by duplications and deletions, and the variable degrees of mosaicism, 45, X cell line in particular, in various tissues. The Aim: This study was conducted to provide more information on patients with idic (Yq) allowing a better phenotype-karyotype correlation and understanding the sexual differentiation in these patients . Patients and Methods: The study included 14 patients referred to the out patient clinic of the Human Genetics Department, Medical Research Institute,University of Alexandria. The reason for referral was genital ambiguity [8 patients], short stature with variable Turner stigmata [4 patients] and primary amenorrhea with normal height [2 patients]. All patients were subjected to clinical examination and chromosome analysis by GTG and CTG-banding techniques. Fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR)were done to determine the structure of the marker chromosomes detected by conventional methods. Results: Chromosome analysis revealed a 45, X/46, X,idic (Y) (p11.3) in ten patients with variable degree of mosaicism, non mosaic 46, X, idic (Y) (p11.3) in two patients and a predominant 46, XX cell line along with 47, XX, idic (Y) (p11.3) cell line in two other patients .While the patients with an idic (Yq) described in this report were phenotypically different, all are considered as being at increased risk for gonadoblastoma. The great phenotypic variations seen in patients with an isodicentric Y chromosome greatly limit the genotype - phenotype correlation. [ABSTRACT FROM AUTHOR]

The article presents a study on the polymorphism in MRL and AKR mice sex-determining region on Y (Sry) genes. It notes that the sex of a mammal is ascertained when an ovum that contains a haploid genome with an X chromosome fertilized by a sperm that has a haploid genome with an X or Y chromosome. The anatomical determination of sex happens toward the development as gonads initially grow as bipotential primordia with plasticity to evolve into ovaries or testes wherein the Sry expression is needed. The study used several processes including the light microscopic analysis, and genomic DNA extraction. Sry PCR and nucleotide sequencing, and comparison of Sry nucleotide sequences and corresponding amino acids between M+ and B6 are also applied.

Therian mammals (marsupials and placentals) have an XX female: XY male sex chromosome system, which is homologous to autosomes in other vertebrates. The testis-determining gene, SRY, is conserved on the Y throughout therians, but is absent in other vertebrates, suggesting that the mammal system evolved about 310 million years ago (MYA). However, recent work on the basal monotreme mammals has completely changed our conception of how and when this change occurred. Platypus and echidna lack SRY, and the therian X and Y are represented by autosomes, implying that SRY evolved in therians after their divergence from monotremes only 166 MYA. Clues to the ancestral mechanism usurped by SRY in therians are provided by the monotremes, whose sex chromosomes are homologous to the ZW of birds. This suggests that the therian X and Y, and the SRY gene, evolved from an ancient bird-like sex chromosome system which predates the divergence of mammals and reptiles 310 MYA. [ABSTRACT FROM AUTHOR]

Transcription factors of the Sox family arose around the advent of multicellularity in animals, arguing that their ability to regulate the expression of extracellular matrix, cell adhesion and signaling molecules may have been instrumental in the generation of metazoans. In particular, during vertebrate evolution, the Sox family experienced a phase of expansion that led to the appearance of groups of highly homologous Sox proteins and the division of existing Sox protein functions among group members. It furthermore allowed Sox transcription factors to acquire numerous novel functions. These past events of subfunctionalization and neofunctionalization can still be recognized today in all groups of the Sox family. They have led to partial functional redundancies, but also to interesting species-specific variations in the developmental roles of Sox proteins as shown here for the SoxB and the SoxE groups. [ABSTRACT FROM AUTHOR]

The reconstruction of evolutionary relationships among a group of closely-related taxa provides a valuable window onto their biogeographic history. This is the first such molecular phylogenetic study to survey all three taxa of the Chlorocebus lhoesti species group, a group of Old World monkeys that includes C. lhoesti, Chlorocebus preussi, and Chlorocebus solatus. Representatives of each species were surveyed for two Y-chromosomal genes, TSPY (∼2240 bp) and SRY (∼780 bp), and one X-chromosomal intergenic region (∼9300 bp) homologous to a portion of human Xq13.3. Maximum likelihood topologies inferred from these sequences confirm that the lhoesti group is monophyletic within the tribe Cercopithecini, as suggested by earlier karyotype studies. Within this group, C. lhoesti (Albertine region) and C. preussi (Cameroon, Nigeria, Bioko) cluster to the exclusion of C. solatus (Gabon). This phylogeographic pattern, evaluated in the context of Pleistocene glacial cycles and lhoesti group ecology, suggests: (1) the common ancestor of the group probably arose in west central Africa, (2) dispersal to the Albertine region is likely to have occurred during a glacial maximum, rather than an interglacial pluvial, and (3) this dispersal probably occurred via a path north of the Congo Basin. Divergence date calculations suggest C. solatus separated from the ancestral stock at ∼2.1 Ma, whereas C. preussi and C. lhoesti bifurcated later, at ∼0.5 Ma. Taken together, these evolutionary analyses draw attention to one period in the Pleistocene (∼0.5 MYA) when a dispersal corridor suitable to montane taxa is likely to have connected the highlands of east central and west central Africa. © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society, 2008, 154, 408–418. [ABSTRACT FROM AUTHOR]

Oligodendrocyte development progresses from specification to terminal differentiation through several phases. By now, a number of transcription factors have been identified that are essential for one or more of these phases. They stem from transcription factor families with known roles in many developmental processes. Basic helix–loop–helix, homeodomain, and high-mobility-group containing transcription factors such as the Olig, Nkx, and Sox proteins have been particularly well studied. A complex picture has emerged in which these transcription factors interact in transcriptional networks and thereby combine and influence their respective activities as repressors or activators in such a way that stage- and cell-type specific gene expression is achieved during oligodendrocyte development. [ABSTRACT FROM AUTHOR]

We describe a simple single-reaction technique for identifying the sex of white-tailed deer (Odocoileus virginianus) based on the PCR amplification of a zinc-finger intron using one pair of primers. Although Sry-coamplification confirmed sex identities, use of the Sry marker was unnecessary due to dimorphic alleles on the X and Y chromosomes at the zinc-finger locus. Insertions in intron 7 of the Y-linked allele (417 bp) make it nearly twice as long as the X-linked allele (236 bp) and thus the amplification products are easily discernable by simple agarose gel electrophoresis. The relatively short size of these products makes them useful for DNA-based sex identification from potentially low-yield tissue samples (e.g., hair, feces). This technique will provide ecologists, conservation geneticists and wildlife managers with a mechanism to readily and reliably identify the sex of unknown white-tailed deer tissue samples, and likely similar samples from other cervid species. [ABSTRACT FROM AUTHOR]

Abstract: A quadruplex real-time qPCR assay was developed to simultaneously assess total human DNA, human male DNA, DNA degradation and PCR inhibitors in forensic samples. Specifically, the assay utilizes a ∼170–190bp target sequence that spans the TH01 STR locus to quantify total human DNA (nuTH01), a 137bp target sequence directly adjacent to the SRY gene to quantify human male DNA (nuSRY), a 67bp target sequence flanking the CSF1PO STR locus (nuCSF) to assess degradation (nuCSF:nuTH01 ratio) and a 77bp synthetic DNA template used as an internal PCR control target sequence (IPC) for the assessment of PCR inhibition. Validation studies, performed on an ABI 7500 SDS instrument using TaqMan® and TaqManMGB® detection, indicate each of the targets in the quadruplex assay performs effectively and is informative even when challenged with DNase-degraded and hematin-inhibited samples. The nuTH01–nuSRY–nuCSF–IPC quadruplex qPCR assay is envisioned to assist in the choice of the most informative DNA typing system available, which may include standard autosomal STR typing when the results indicate the presence of non-degraded, single gender DNA or non-degraded, male:female mixtures at ratios expected to yield probative alleles; Y STR typing in samples containing a male component that is overwhelmed by the presence of an excess of female DNA; reduced amplicon size STR typing (“MiniSTRs”) where the nuCSF:nuTH01 ratio indicates the sample is highly degraded; enhanced STR amplification with additional AmpliTaq Gold®/BSA and/or sample clean-up when the presence of PCR inhibitors is suggested by a delayed IPC C T value or mitochondrial DNA typing in samples where little to no nuclear DNA is detected. The present study includes evaluations of species specificity, sensitivity, precision, reproducibility, male–female mixtures, population samples and applications to various casework-type samples as indicated by the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. [Copyright &y& Elsevier]

Abstract: This report details a chimeric goat derived by injecting caprine embryonic germ (EG) cells into a host blastocyst. The EG cells, isolated from the primordial genital ridge of white Guanzhong goat fetuses (28–42 days of pregnancy), had alkaline phosphatase activity and several stem cell markers, including SSEA-1, c-kit, and Nanog. Ten to 20EG cells were microinjected into the blastocoelic cavity of a host blastocyst collected from a black goat following natural service. Twenty-nine injected blastocysts were transferred into nine white surrogate goats. One of the recipients maintained pregnancy to term and gave birth to three kids: one male, one female, and a dead, malformed fetus of undetermined gender; all three fetuses were black, but the female and the malformed fetus each had a large white spot on their head. Based on PCR and microsatellite DNA assay, the female and the malformed fetus were monozygotic twins and chimeras. Microsatellite assay on various tissues from the dead fetus (including skin, blood, liver, placenta, lung, heart, spleen, muscle, and brain), revealed that these tissues and organs were chimeric and contained cells derived from EG cells. In conclusion, caprine EG cells differentiated into all three germ layers in vivo. [Copyright &y& Elsevier]

Recent studies have shown significant sex differences in the pattern of 2D:4D finger length ratios in humans and several other mammalian species. In humans, these ratios are suggested to be negatively correlated with prenatal exposure to testosterone, positively correlated with prenatal estrogen, and exhibit sex specific patterns of association with sexually dimorphic clinical phenotypes. However, the relative contributions of genetic and environmental influences on digit ratios in men and women are currently unknown. Therefore, the purpose of the current study was to examine genetic and environmental influences on 2D:4D ratios in twins. Participants included 146 monozygotic (MZ) and 154 dizygotic (DZ) adult male and female twins participating in the Michigan State University Twin Study of Behavioral Adjustment and Development. Overall, biometric model-fitting analyses indicated significant additive genetic and nonshared environmental influences on digit ratios. Findings suggest greater similarity between 2D:4D ratios in MZ relative to DZ twins that can be accounted for by genetic and nonshared environmental factors. [ABSTRACT FROM AUTHOR]

Studies on demographic population history and gene flow among populations often rely exclusively on matrilinearly inherited mitochondrial DNA markers. However, by excluding patrilines, such approach introduces an analytical bias into the study. To overcome this bias, we established a set of ten Y-chromosomal markers for the European brown hare ( Lepus europaeus), which comprises of three overlapping fragments spanning over the sex-determining region Y, five microsatellite loci ( LeMS-Y), and two introns of the Y-linked zinc finger protein ( LeZFY). Besides the generation of male specific fragments, both the ZFY and the LeMS-Y01 primer pairs also generated amplification products in females, which are visible in standard agarose gels. These polymerase chain reaction (PCR) products were easily distinguishable from the Y-specific amplicons and thus can function as internal positive PCR control in molecular sexing. [ABSTRACT FROM AUTHOR]

Previous reports suggested that humans and mice differ in their sensitivity to the genetic dosage of transcription factors that play a role in early testicular development. This difference implies that testis determination might be somewhat different in these two species. We report that the Fog2 and Gata4 transcription factors are haploinsufficient for testis determination in mice. Whether gonadal sex reversal occurs depends on genetic background (i.e., modifier genes). For example, C57BL/6J (B6) XY mice develop testes if they are heterozygous for a mutant Fog2 (Fog2-) or Gata4 (Gata4ki) allele. However, if the B6 Y chromosome (YB6) is replaced by the AKR Y chromosome (YAKR). B6 Fog2-/+ XYAKR mice develop ovaries, and B6 Gata4ki/+ XYAKR mice develop ovaries and ovotestes (gonads containing both ovarian and testicular tissue). Furthermore, DBA/2J (D2) Fog2-/+ XYAKR mice and (B6 x D2)F1 hybrid Gata4ki/+ XYAKR mice develop testes. Sry is expressed in the mutant XY gonads, indicating that the lack of Sry expression is not the cause of ovarian tissue development in B6 Fog2-/+ or Gata4ki/+ XYAKR mice. However, up-regulation of Sox9 expression, which is critical for normal testicular development, does not occur in mutant XY gonads that develop as ovaries. We conclude that under certain genetic conditions, Sox9 up-regulation depends on the proper dosage of Fog2 and Gata4. We propose that in humans the FOG2 and/or GATA4 genes might be haploinsufficient for normal testis determination and thus could be the cause of some previously unassigned cases of XY gonadal sex reversal. [ABSTRACT FROM AUTHOR]

Abstract: Sex determination in vertebrates is accomplished through a highly conserved genetic pathway. But surprisingly, the downstream events may be activated by a variety of triggers, including sex determining genes and environmental cues. Amongst species with genetic sex determination, the sex determining gene is anything but conserved, and the chromosomes that bear this master switch subscribe to special rules of evolution and function. In mammals, with a few notable exceptions, female are homogametic (XX) and males have a single X and a small, heterochromatic and gene poor Y that bears a male dominant sex determining gene SRY. The bird sex chromosome system is the converse in that females are the heterogametic sex (ZW) and males the homogametic sex (ZZ). There is no SRY in birds, and the dosage-sensitive Z-borne DMRT1 gene is a credible candidate sex determining gene. Different sex determining switches seem therefore to have evolved independently in different lineages, although the complex sex chromosomes of the platypus offer us tantalizing clues that the mammal XY system may have evolved directly from an ancient reptile ZW system. In this review we will discuss the organization and evolution of the sex chromosomes across a broad range of mammals, and speculate on how the Y chromosome, and SRY, evolved. [Copyright &y& Elsevier]

Abstract: In this report we present a case of the male-to-female sex reversal syndrome in a Dachshund bitch. The animal was subjected for examination due to the enlargement of clitoris and male-type behaviour. The cytogenetic analysis (Giemsa staining and FISH with sex chromosome specific probes) revealed a normal male chromosome complement (78,XY). The molecular analysis of the SRY and SOX9 genes with the use of SSCP technique did not reveal any polymorphism. Blood testosterone concentration was low (0.75ng/ml), thus the androgen insensitivity syndrome could be excluded. We suggest that this case may represent a persistent Müllerian duct syndrome. [Copyright &y& Elsevier]

Abstract: The first objective was to correlate calving sex-ratio data from semen lots with the semen sex ratio obtained by two duplex polymerase chain reaction (PCR)/gel electrophoresis techniques. The two techniques involved different starting DNA amounts, PCR conditions, agarose gel concentrations, sample placement on the gels, lane size, number of lanes per gel, and duration of electrophoresis. The second objective was to sequence the duplex PCR products to verify their match to genes and chromosomes for which they were designed. Thirty-six ejaculates (lots) from eight Holstein sires were collected. Semen straws were distributed among dairies in three states. Ten straws per lot were used for the different PCR techniques. Sperm DNA was extracted and PCR analysis was done using one primer set to amplify a single copy section of the factor IX precursor (X-chromosome only) and another primer set to amplify a single copy section the sex determining region (Y-chromosome only). The glyceraldehyde phosphate dehydrogenase gene was amplified as an internal control. Standard curves were designed using PCR products in known ratios. Gel electrophoresis and image analysis were used to determine predicted %Y-chromosome-bearing spermatozoa (PredPtY). Sex (male=1, female=0) was reported on 526 calves and the ratio of the number of male to total calves (proportion of male calves (PMC)) was determined between sire and lot within sire. The PredPtY and PMC were significantly correlated (r =0.82, P <0.0002). No significant variance between sires was found in PredPtY or PMC, but lots within sires was a significant variance source for both. The two PCR technologies adequately determined semen sex ratio. The technology-by-lot-within-sire interaction was a significant variance source for PredPtY. Acrosomal integrity (after a 2-h) incubation, was correlated with both PMC and PredPtY; other semen quality characteristics had no significant correlations with PMC or PredPtY. Therefore, calf crop sex ratio skewness could be controlled by screening semen for PredPtY through the use of PCR. [Copyright &y& Elsevier]

Abstract: In mammals, Sry (sex-determining region Y gene) is the master regulator of male sex determination. The discovery of Sry in 1990 was expected to provide the key to unravelling the network of gene regulation underlying testis development. Intriguingly, no target gene of SRY protein has yet been discovered, and the mechanisms by which it mediates its developmental functions are still elusive. What is clear is that instead of the robust gene one might expect as the pillar of male sexual development, Sry function hangs by a thin thread, a situation that has profound biological, medical and evolutionary implications. [Copyright &y& Elsevier]

Abstract: Males and females of many species differ with regard to neurodevelopment, ongoing brain function and behavior. For many years, it was assumed that these differences primarily arose due to hormonal masculinization of the male brain (and to a lesser extent hormonal feminization of the female brain). Recent elegant experiments in model systems have revealed that, while gonadal hormones undoubtedly play an important role in sexual differentiation of the brain, they are not the only possible mechanism for this phenomenon. In the present review, we discuss the concept that genes residing upon the sex chromosomes (which are asymmetrically inherited between males and females) may influence sexually dimorphic neurobiology directly, and suggest possible mechanisms. Future work will be directed towards understanding the extent and specificity with which sex-linked genes and hormones define brain structure and function, and towards elucidating potential interactions between the two mechanisms. Ultimately, it is hoped that such studies will provide insights into why men and women are differentially vulnerable to certain mental disorders, and will enable the development of effective sex-tailored therapeutics. [Copyright &y& Elsevier]

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Abstract: In the central nervous system, Connexin32 and Connexin47 are confined to oligodendrocytes where they contribute to myelin formation and maintenance, and are essential for establishing a functional glial syncytium that ensures ionic homeostasis. Despite their importance, not much is known about the regulation of connexin gene expression in oligodendrocytes. Here, we identify group E Sox proteins, in particular Sox10, as essential transcriptional regulators of both connexins. Not only was expression of Connexin32 and Connexin47 severely compromised in spinal cords of mouse mutants with reduced amounts of group E Sox proteins. Sox10 also stimulated in transient transfections the Connexin32 promoter as well as Connexin47 promoter 1b which is the main Connexin47 promoter active in the postnatal spinal cord. Detailed characterization of Connexin47 promoter 1b identified a single monomer binding site that mediated Sox10-dependent promoter activation. The region containing this binding site was also occupied by endogenous Sox10 in 33B oligodendroglioma cells. These results add Connexin47 and Connexin32 to the list of Sox10 target genes and argue that Sox10 may influence transcription of many terminal differentiation and myelination genes in oligodendrocytes as an essential regulatory component of the myelination program. [Copyright &y& Elsevier]

Abstract: The high-mobility group protein HMGB1 contains two tandem DNA-binding HMG box domains, A and B, linked by a short flexible linker that allows the two domains to behave independently in the free protein. There is no structural information on how the linked domains and linker behave when bound to DNA, mainly due to the lack of any DNA-sequence preference of HMGB1. We report the structure determination, by NMR spectroscopy, of a well-defined complex of two tandem HMG boxes bound to a 16 bp oligonucleotide. The protein is an engineered version of the AB di-domain of HMGB1, in which the A box has been replaced by the HMG box of the sequence-specific transcription factor SRY, to give SRY.B. In the SRY.B/DNA complex, both HMG boxes bind in the minor groove and contribute to the overall DNA bending by intercalation of bulky hydrophobic residues between base-pairs; the bends reinforce each other, and the basic linker lies partly in the minor groove. As well as being the first structure of an HMG-box di-domain bound to DNA, this provides the first structure of the B domain of HMGB1 bound to DNA. [Copyright &y& Elsevier]

Abstract: The testis-determining gene SRY is not well-conserved among mammals, particularly between mouse and other mammals, both in terms of protein structure and of expression regulation. To evaluate SRY phylogenic conservation in regards to its function, we expressed the goat gene (gSRY) in XX transgenic mouse gonads. Here, we show that gSRY induces testis formation, despite a goat expression profile. Our results demonstrate that sex-reversal can be induced in XX-mice by a non-mouse SRY thus suggesting a conserved molecular mechanism of action of this testis-determining gene across mammalian species. [Copyright &y& Elsevier]

Abstract: Among the structural abnormalities affecting the human Y chromosome, dicentric chromosomes are the most common. A wide spectrum of phenotypes of patients with a dicentric Y chromosome exists, ranging from almost males through mixed gonadal dysgenesis to females with Turner syndrome. Here, we describe an infant with mixed gonadal dysgenesis and mosaic karyotype 45,X/46,X,idic(Y)(qter→p11.32:p11.32→qter)/47,X,+2idic(Y) (qter→p11.32:p11.32→qter)/47,XYY. This was demonstrated by fluorescence in situ hybridization (FISH) analysis with whole Y chromosome painting (WCP-Y) probe. Molecular studies were performed on genomic DNA extracted from peripheral blood lymphocytes. To examine the sex determined region (SRY), azoospermia factor (AZF) region and deletion in azoospermia gene (DAZ), polymerase chain reaction (PCR) analyses were done with sequence-tagged site (STS) primers of 20 loci along the Y chromosome (SRY, DYS271, DYS148, DYS273, KALY, DYS212, SMCY, DYS215, DYS218, DYS219, DYS221, DYS223, DYS224, DYF51S1, DYS236, DAZ, DYS240), and all tested loci were found positive. Because of the possibility of a mutation in the SRY gene, we analyzed the PCR fragment by DNA sequencing and did not observe any mutation or nucleotide alteration. We present detailed molecular-cytogenetic characterization of a patient with idic(Y)(p11.32), and results are discussed with the previously described patients. As far as we know, this is the fifth report of a 46,X, idic(Y)(p11.32) karyotype and the first presentation with mixed gonadal dysgenesis and isodicentric Y. Since the correlation between phenotype and karyotype is not yet well defined, the clinical reports will be helpful in defining the phenotypic range of this chromosomal abnormality. [Copyright &y& Elsevier]

Abstract: We have successfully specified essential sequences of the 5′ upstream region for the stage- and tissue-specific expression of mouse Sry by using an in vitro Cre/loxP system. Sry/Cre plasmids carrying Sry 5′ sequences of various sizes were transfected into the primary cultured cells from different tissues of CAG/loxP/CAT/loxP/LacZ transgenic fetuses on 11.5-day post coitus (dpc) or 13.5-dpc. Stage- and tissue-specific regulation of Sry expression was disrupted by the deletion of positions 7549–7660 (from −0.4 to −0.5kb region). In vitro transcription assay also suggested that the region contains element(s) responsible for stage- and tissue-specific expression of mouse Sry. SRY promoter of Shiba goat (Capra hircus var Shiba), a native Japanese miniature goat, showed the tissue-specific activity in the cells from urogenital ridges of the male mouse, but not in the cells from female mice, indicating a possibly different mechanism among species in the regulation of Sry expression. [Copyright &y& Elsevier]

The high-mobility group transcription factor Sox10 exerts many different roles during development of the neural crest and nervous system. To unravel its complex transcriptional functions, we have started to look for interaction partners. Here, we identify an association of Sox10 with the N-myc interactor Nmi, which was mediated by the high-mobility group of Sox10 and the central region of Nmi. In vivo relevance of this interaction is indicated by the fact that both proteins were co-expressed in glial cells, gliomas and in the spinal cord. Additionally, subcellular localization of Nmi in C6 glioma depended on the presence of Sox10 such that nuclear Nmi was more frequent in Sox10-expressing cells. Importantly, Nmi modulated the transcriptional activity of Sox10 in reporter gene assays. Nmi effects varied between different Sox10 target gene promoters, indicating that Nmi function in vivo may be promoter-specific. [Copyright &y& Elsevier]

Abstract: We have raised an antibody specifically recognizing endogenous mouse SRY protein and used it to investigate the molecular and cellular mode of action of SRY in testis determination. We find that expression of SRY protein closely mirrors the expression of Sry mRNA in mouse genital ridges and is detectable for 6 to 8 h after the mRNA ceases to be detectable. The subset of somatic cells that expresses SRY begins to express SOX9 almost immediately. Since these SOX9-positive cells go on to develop as Sertoli cells, it appears that SRY expression marks the pre-Sertoli cell lineage and leads to up-regulation of Sox9 expression cell-autonomously. However, a small proportion of SOX9-positive cells did not appear to express SRY, possibly reflecting the additional involvement of paracrine signaling in activating Sox9 transcription in these cells. We confirmed by ex vivo cell mixing experiments that SRY is able to engage receptor-mediated signaling to up-regulate Sox9 expression. Finally, we showed by employing specific inhibitors that the causative signaling molecule is prostaglandin D2 (PGD2) and that PGD2 can induce Sox9 transcription in cultured XX gonads. Our data indicate a mechanism whereby Sry uses both a cell-autonomous mechanism and a PGD2-mediated signaling mechanism to stimulate expression of Sox9 and induce the differentiation of Sertoli cells in vivo. [Copyright &y& Elsevier]

Abstract: Male-specific migration of cells from the mesonephric kidney into the embryonic gonad is required for testis formation in the mouse. It is unknown, however, whether this process is specific to the mouse embryo or whether it is a fundamental characteristic of testis formation in other vertebrates. The signalling molecule/s underlying the process are also unclear. It has previously been speculated that male-specific cell migration might be limited to mammals. Here, we report that male-specific cell migration is conserved between mammals (mouse) and birds (quail–chicken) and that it involves proper PDGF signalling in both groups. Interspecific co-cultures of embryonic quail mesonephric kidneys together with embryonic chicken gonads showed that quail cells migrated specifically into male chicken gonads at the time of sexual differentiation. The migration process is therefore conserved in birds. Furthermore, this migration involves a conserved signalling pathway/s. When GFP-labelled embryonic mouse mesonephric kidneys were cultured together with embryonic chicken gonads, GFP+ mouse cells migrated specifically into male chicken gonads and not female gonads. The immigrating mouse cells contributed to the interstitial cell population of the developing chicken testis, with most cells expressing the endothelial cell marker, PECAM. The signalling molecule/s released from the embryonic male chicken gonad is therefore recognised by both embryonic quail and mouse mesonephric cells. A candidate signalling molecule mediating the male-specific cell migration is PDGF. We found that PDGF-A and PDGF receptor-α are both up-regulated male-specifically in embryonic chicken and mouse gonads. PDGF signalling involves the phosphotidylinositol 3-kinase (PIK3) pathway, an intracellular pathway proposed to be important for mesonephric cell migration in the mammalian gonad. We found that a component of this pathway, PI3KC2α, is expressed male-specifically in developing embryonic chicken gonads at the time of sexual differentiation. Treatment of organ cultures with the selective PDGF receptor signalling inhibitor, AG1296 (tyrphostin), blocked or impaired mesonephric cell migration in both the mammalian and avian systems. Taken together, these studies indicate that a key cellular event in gonadal sex differentiation is conserved among higher vertebrates, that it involves PDGF signalling, and that in mammals is an indirect effect of Sry expression. [Copyright &y& Elsevier]

The nuclear receptor transcription factor Dax1 is hypothesized to play a role in testicular development, although the mechanism of its action is unknown. Here, we present evidence that Dax1 plays an early essential role in fetal testis development. We hypothesize that upregulation of Sox9 expression in precursor somatic cells, a process required for their differentiation as Sertoli cells, depends on the coordinated expression of Dax1, Sry and another gene, Tda1. Our conclusion and model are based on the following experimental findings: (1) presence of a mutant Dax1 allele (Dax1-) results in complete gonadal sex reversal in C57BL/6JEi (B6) XY mice, whereas testes develop in DBA/2J (D2) and (B6xD2)F1 XY mice; (2) B6-DAX1 sex reversal is inherited as a complex trait that includes the chromosome 4 gene Tda1; (3) B6 Dax1-/Y fetal gonads initiate development as ovaries, even though Sry expression is activated at the correct time and at appropriate levels; (4) upregulation of Sox9 does not occur in B6 Dax1-/Y fetal gonads in spite of apparently normal Sry expression; and (5) overexpression of Sry in B6 Dax1-/Y fetal gonads upregulates Sox9 and corrects testis development. [ABSTRACT FROM AUTHOR]

Sry (sex-determining region on the Y chromosome) is a master gene that initiates testis differentiation of the bipotential indifferent gonad in mammals. In mice, Sry expression is transiently activated in a center-to-pole wave along the anteroposterior (AP) axis of developing XY gonads. Shortly after the onset of Sry activation, Sox9 (Sry-related HMG box-9), a fundamental testis-differentiation gene common to all vertebrates, is also activated in a center-to-pole pattern similar to the initial Sry expression profile. Several male-specific cellular events, such as glycogenesis, coelomic epithelium proliferation, mesonephric migration and vasculogenesis, are induced in XY gonads following the onset of Sry and Sox9 expression. This paper mainly focuses on recent advances in elucidating the regulatory mechanisms of Sry and Sox9 expression and male-specific cellular events immediately downstream of SRY action during the initial phases of testis differentiation. [ABSTRACT FROM PUBLISHER]

Abstract: The myelin-forming oligodendrocytes of the mouse embryonic spinal cord express the three group E Sox proteins Sox8, Sox9, and Sox10. They require Sox9 for their specification from neuroepithelial cells of the ventricular zone and Sox10 for their terminal differentiation and myelination. Here, we show that during oligodendrocyte development, Sox8 is expressed after Sox9, but before Sox10. Loss of Sox8 did not impair oligodendrocyte specification by itself, but enhanced the Sox9-dependent defect. Oligodendrocyte progenitors were still generated in the Sox9-deficient spinal cord, albeit at 20-fold lower rates than in the wildtype. Combined loss of Sox8 and Sox9, in contrast, led to a near complete loss of oligodendrocytes. Other cell types such as ventricular zone cells and radial glia remained unaffected in their numbers as well as their rates of proliferation and apoptosis. Oligodendrocyte development thus relies on the differential contribution of all three group E Sox proteins at various phases. [Copyright &y& Elsevier]

Sry is transiently activated in pre-Sertoli cells of the gonadal ridge to initiate testis differentiation in mice. In pre-Sertoli cells, however, the cellular events induced immediately after the onset of Sq expression remain largely unknown. Here we show that testis-specific glycogen accumulation in pre-Sertoli cells is one of the earliest cellular events downstream of Sq action. In developing XY gonads, glycogen accumulation starts to occur in pre-Sertoli cells from around 11.15 dpc (tail somite 14 stage) in a center-to-pole pattern similar to the initial Sq expression profile. Glycogen accumulation was also found in XX male gonads of Sry-transgenic embryos, but not in XX female gonads of wildtype embryos at any developmental stage. In vitro analyses using various culture conditions suggest that testis-specific glycogen deposition is a tissue-autonomous event that can be induced even in serum-free conditions and in a culture of gonadal explants without adjacent mesonephros. Moreover, glycogen accumulation in pre-Sertoli cells was significantly inhibited in vitro by the PI3K inhibitor LY294002, but not by the MEK inhibitor PD98059. Active phospho-AKT (PI3K effector) showed a high degree of accumulation in gonadal somatic cells of genital ridges in a testis-specific manner, both in vitro and in vivo. Therefore, these findings suggest that immediately after the onset of Sry expression, activation of the PI3K-AKT pathway promotes testis- specific glycogen storage in pre-Sertoli cells. To the best of our knowledge, this is a novel Sty-downstream cellular event which preserves this readily available energy source in Sertoli cells for testis-specific morphogenesis and hormone production. [ABSTRACT FROM AUTHOR]

Sox proteins are transcriptional regulators with a high-mobility-group domain as sequence-specific DNA-binding domain. For function, they generally require other transcription factors as partner proteins. Sox proteins furthermore affect DNA topology and may shape the conformation of enhancer-bound multiprotein complexes as architectural proteins. Recent studies suggest that Sox proteins are tightly regulated in their expression by many signalling pathways, and that their transcriptional activity is subject to post-translational modification and sequestration mechanisms. Sox proteins are thus ideally suited to perform their many different functions as transcriptional regulators throughout mammalian development. Their unique properties also cause Sox proteins to escape detection in many standard transcription assays. In melanocytes, studies have so far focused on the Sox10 protein which functions both during melanocyte specification and at later times in the melanocyte lineage. During specification, Sox10 activates theMitfgene as the key regulator of melanocyte development. At later stages, it ensures cell-type specific expression of melanocyte genes such asDopachrome tautomerase. Both activities require cooperation with transcriptional partner proteins such as Pax-3, CREB and eventually Mitf. If predictions can be made from other cell lineages, further functions of Sox proteins in melanocytes may still lie ahead. [ABSTRACT FROM AUTHOR]

Abstract: The phenomenon of B6-YDOM sex reversal arises when certain variants of the Mus domesticus Y chromosome are crossed onto the genetic background of the C57BL/6J (B6) inbred mouse strain, which normally carries a Mus musculus-derived Y chromosome. While the sex reversal has been assumed to involve strain-specific variations in structure or expression of Sry, the actual cause has not been identified. Here we used in situ hybridization to study expression of Sry, and the critical downstream gene Sox9, in strains containing different chromosome combinations to investigate the cause of B6-YDOM sex reversal. Our findings establish that a delay of expression of SryDOM relative to SryB6 underlies B6-YDOM sex reversal and provide the first molecular confirmation that Sry must act during a critical time window to appropriately activate Sox9 and effect male testis determination before the onset of the ovarian-determining pathway. [Copyright &y& Elsevier]

Abstract: Testis induction is associated with gonadal Sry and Sox9 expression in mammals. This study investigated whether Sry expression directly induces male-specific Sox9 activation during early phases of testis differentiation. We have established an XX sex-reversal mouse line carrying the Sry transgene driven by a weak basal promoter of the Hsp70.3 gene (Hsp-Sry), whereby the transgene was activated in the gonads along the entire anteroposterior axis from earlier stages. The effects of misexpression and overexpression of Sry on the spatiotemporal pattern of Sox9 expression were examined using both XX and XY gonads of Hsp-Sry transgenic embryos. It was shown that ectopic expression of Sry transcripts in the entire gonadal area from earlier stages promotes neither any advance in the timing nor any appreciable ectopic activation of endogenous Sox9 expression. Immediately after the onset of Sox9 activation, however, both the level of Sox9 expression and the number of SOX9-positive cells were significantly enhanced in Hsp-Sry/XY gonads, as compared with those in wild-type/XY and Hsp-Sry/XX gonads. These findings suggest that, although Sry is capable of up-regulating Sox9 expression dose-dependently, Sry mRNA expression alone is not likely to provide positional or timing information needed for male-specific Sox9 activation in developing XY gonads. [Copyright &y& Elsevier]