Testis-determining Factor Gene Research Articles

Effects of tamoxifen on autosomal genes regulating ovary maintenance in adult mice.

Environmental endocrine-disrupting chemicals (EDCs), known to bind to estrogen/androgen receptors and mimic native estrogens, have been implicated as a main source for increasing human reproductive and developmental deficiencies and diseases. Tamoxifen (TAM) is one of the most well-known antiestrogens with defined adverse effects on the female reproductive tract, but the mechanisms related to autosomal gene regulation governing ovary maintenance in mammals remain unclear. The expression pattern and levels of key genes and proteins involved in maintaining the ovarian phenotype in mice were analyzed. The results showed that TAM induced significant upregulation of Sox9, which is the testis-determining factor gene. The results showed that TAM induced significant upregulation of Sox9, the testis-determining factor gene, and the expression level of Sox9 mRNA in the ovaries of mice exposed to 75 or 225 mg/kg bw TAM was 2- and 10-fold that in the control group, respectively (p < 0.001). Furthermore, the testicular fibroblast growth factor gene, Fgf9, was also elevated in TAM-treated ovaries. Accordingly, expression of the ovary development marker, forkhead transcription factor (FOXL2), and WNT4/FST signaling, were depressed. The levels of protein expression changed consistently with the target genes. Moreover, the detection of platelet/endothelial cell adhesion molecule 1 (PECAM-1) in TAM-treated ovaries suggested the formation of vascular endothelial cells, which is a further evidence for the differentiation of the ovaries to a testis-like phenotype. During this period, the level of 17β-estradiol, progesterone, and luteinizing hormone decreased, while that of testosterone increased by 3.3-fold (p = 0.013). The activation of a testis-specific molecular signaling cascade was a potentially important mechanism contributing to the gender disorder induced by TAM, which resulted in the differentiation of the ovaries to a testis-like phenotype in adult mice. Limited with a relatively higher exposure, the present study provided preliminary molecular insights into the sexual disorder induced by antiestrogens and compounds that interrupted estrogen signaling by other modes of action.

H-Y antigens as Y chromosome-encoded gene products and serologically detectable male antigens (SDM) as testis- or spermatogenesis-linked autosomal gene products

Abstract. Male-specific transplantation antigen H-Y was proposed to be the testis-determining factor (TDF) in 1975, while SRY was found to be the TDF gene in 1990. What then of H-Y antigen? H-Y antigen was categorized into two entities, viz., T-cell mediated H-Y antigens (H-Y) and serologically detectable male antigens (SDM). Several HY genes such as Smcy, Uty and Dty have been identified and these are all Y-Iinked. H-Y is male-specific and clinically important in cell, tissue, or organ transplantations. Male-enhanced antigen 1 (Meal) was isolated from an expression library using polyclonal anti-H-Y antibody. We cloned and characterized mouse and bovine Meal/MEAI, the gene product of which was mainly localized in elongated spermatids. Mea2 was identified using monoclonal anti-H-Y antibody. MEA2 protein is localized in the Golgi apparatus of spermatocytes and spermatids. Colocalization of MEA2 protein with y-adaptin in clathrin-coated vesicles was demonstrated. Disruption of Mea2 resulted in spermatogenic failure. These findings suggest that Mea2 is involved in transportation of materials needed for acrosome components in spermatogenesis. Its human homologue is Golgin-160 which was detected in an SLE autoimmune disease patient. Müllerian inhibiting substance (MIS) was reported to have SDM activity. Taken together, SDM is a collective name for protein antigens associated with testis activity (MIS) or spermatogenesis (MEA1, MEA2) and may be antigenic when expressed in females. From the viewpoint of autoimmune diseases, the Identification and characterization of SDMs will be clinically important.

Members of the SRY family regulate the human LINE retrotransposons.

LINEs are endogenous mobile genetic elements which have dispersed and accumulated in the genomes of most higher eukaryotes via germline transposition, with up to 100 000 copies for the human LINE-1 (L1H) sequences. Although severely repressed in most normal tissues, L1H is still functional, with evidence for both germline and somatic-essentially in tumors-transpositions. Yet, no transcription factor that could regulate their transcription and be responsible for their transposition has hitherto been described. Here we show that factors belonging to the family of the testis-determining factor gene SRY (the SOX family) can modulate L1H promoter activity over a 10-fold range in a transient transfection assay using a luciferase reporter gene. These effects depend on two functional SRY binding sites which can be identified within the L1H promoter via mobility shift assays. Induction of endogenous L1Hs upon ectopic expression of the SOX11 transcription factor is further demonstrated, thus strengthening the physiological relevance of these new-and highly dispersed-target sites for the otherwise unclassical transcription factors of the SRY family.

Peter Goodfellow: sex, drugs and poetry

Peter Goodfellow: sex, drugs and poetry

Isodicentric Y chromosome: cytogenetic, molecular and clinical studies and review of the literature

Dicentrics are among the most common structural abnormalities of the human Y chromosome. Predicting the phenotypic consequences of different duplications and deletions of dicentric Y chromosomes is usually complicated by varying degrees of mosaicism (45,X cell lines), which may, in some cases, remain undetected. Molecular studies in patients with dicentric Y chromosomes have been few, and only two studies have attempted to determine the presence of SRY (the putative testis-determining factor gene). We report an 18-year-old female with short stature, amenorrhea, hirsutism, hypoplastic labia minora, and clitoromegaly who has a 45,X/46,X,idic(Y)(p11.32)/47,X,idic(Y)(p11.32),idic(Y) (p11.32) karyotype. Southern analysis using Y-specific probes (Y97, 2D6, 1F5, pY3.4) and polymerase chain reaction (PCR) analysis using primers for ZFY and SRY were positive for all loci tested, indicating that almost all of the Y chromosome was present. Our findings and an extensive review of the literature emphasize the importance of molecular analyses of abnormal Y chromosomes before any general conclusions can be reached concerning the relative effects of the Y-chromosome abnormality and mosaicism on sexual differentiation.

Characterization and sequencing of the sex determining region Y gene (Sry) in Akodon (Cricetidae) species with sex reversed females.

The sex determining region Y gene (Sry) is the strongest candidate to be the testis determining factor gene (Tdy). Several South-American Akodon species have two varieties of Y chromosome. One type transmitted via male specimens induces testis development. The second Y variety fails to induce male gonadal differentiation and gives rise to fully fertile XY females. These variant females test positive for Sry. Moreover, sequencing of a partial open reading frame of the conserved region of Sry from males and XY females shows no sequence difference. Sry is two- to sixfold amplified in six of eight akodont species tested. Since Sry amplification was found in species having and not having XY females, amplification apparently does not in itself play a primary role in the origin of sex reversal. The development of fully fertile ovaries in XY Akodon females is not due to a deletion of Sry or to mutations in the Sry segment analyzed in this report. Sex reversal may be due to abnormal expression of this gene at the stage of gonadal differentiation. Alternatively, other genes in the sex-determining pathway may be involved. Several of the Akodon species showing Sry amplification also have amplification of Zfy, which may map to the same region of the Akodon Y chromosome.

Sequences Homologous to the Human X- and Y-Borne Zinc Finger Protein Genes (ZFX/Y) Are Autosomal in Monotreme Mammals

The human zinc finger protein genes (ZFX/Y) were identified as a result of a systematic search for the testis-determining factor gene on the human Y chromosome. Although they play no direct role in sex determination, they are of particular interest because they are highly conserved among mammals, birds, and amphibians and because, in eutherian mammals at least, they have active alleles on both the X and the Y chromosomes outside the pseudoautosomal region. We used in situ hybridization to localize the homologues of the zinc finger protein gene to chromosome 1 of the Australian echidna and to an equivalent position on chromosomes 1 and 2 of the platypus. The localization to platypus chromosome 1 was confirmed by Southern analysis of a Chinese hamster X platypus cell hybrid retaining most of platypus chromosome 1. This localization is consistent with the cytological homology of chromosome 1 between the two species. The zinc finger protein gene homologues were localized to regions of platypus chromosomes 1 and 2 that included a number of other genes situated near ZFX on the short arm of the human X chromosome. These results support the hypothesis that many of the genes located on the short arm of the human X were originally autosomal and have been translocated to the X chromosome since the eutherian-metatherian divergence.

Effect on embryos of injection of phosphorothioate-modified oligonucleotides into pregnant mice.

Phosphorothioate-modified oligonucleotides were injected into pregnant female mice to assess the effect on developing embryos. Injections were carried out during two different time periods, one when embryos were in preimplantation stages of development (about 3.5 days of development) and the other after implantation, when both a fetus and placenta are present (from days 9.5 to 11.5 of development). Three different phosphorothioate-modified oligonucleotides were injected. One, which had a sequence not present in the mouse genome, was used to ask whether nonspecific toxic or teratogenic effects on embryos result from treatment of the mother. A second was complementary to the mRNA of the testis-determining factor gene Sry and was used to ask whether a specific developmental pathway (i.e., sex determination) could be disrupted in embryos in vivo. The third was the complement of the anti-Sry sequence. None of these oligonucleotides reduced the frequency of successful pregnancy after mating or the average litter size from that observed in controls animals. Furthermore, examination of 291 pups or fetuses from all oligonucleotide-injected pregnant females revealed no developmental defects regardless of which sequence was used. It is concluded that injection of phosphorothioate-modified oligonucleotides into pregnant females according to the protocols described here is not toxic or teratogenic to embryos in a nonspecific way. Also, anti-Sry oligonucleotides did not influence sex determination in embryos, although there are several possible explanations for this.

Abnormalities of gonadal determination and differentiation.

In the first several weeks of gestation, a variety of bipotential and neutral sexual structures form. These include the bipotential gonadal ridge, a paired set of Wolffian and Mullerian ducts, and the neutral external genitalia.’ With appropriate signals, these structures differentiate to form male or female sex organs.’ The role of the Y chromosome in male sex determination is well established.3-5 The gene on the Y chromosome that is responsible for initiating testis development is called the testis-determining factor (TDF). As discussed earlier, current evidence indicates that the “sex-determining region Y” gene (SRY) is identical to the TDF gene.“,7 The term “gonadal dysgenesis” was coined originally before the advent of karyotype analysis to describe all subjects with female genitalia, normal Mullerian structures, and streak gonads. While many of these subjects had the typical features of Turner syndrome, others had none. The condition of patients without Turner stigmata has been referred to as “pure gonadal dysgenesis”.15,16 When karyotype analysis became available, it was observed that the majority of patients with “gonadal dysgenesis” have a 45,X karyotype or a variant that we now consider part of the spectrum of Turner syndrome.17 Ovarian determination and subsequent steps of sex differentiation are considered to be normal in these subjects. However, subjects with 45,X Turner syndrome have accelerated loss of germ cells and premature degeneration of the ovary.‘*

Testis-determining factor and Y-linked sex reversal

A gene named SRY, isolated last year from the sex-determining region of the human Y chromosome, satisfies many of the criteria expected of the testis-determining factor gene. Mutations in SRY have been found in XY females, strongly implicating SRY as the testis-determining gene.

Sex Determination in Mammals. How Many Genes are Involved?1

The genetic mechanisms of sex determination in mammals have not yet been clarified. Y-linked and X-linked zinc finger genes from humans and mice were recently cloned and characterized. The Y-linked zinc finger gene was originally thought to be the testis-determining factor. However, at the present time, it seems clear that this gene is not the master gene triggering the cascade of events leading to sex determination. The human testis-determining factor gene is known to be located in the nonhomologous region of the short arm of the Y chromosome, close to an Alu repeat that marks the boundary of the pseudoautosomal region. Lately, this region has been found to contain a new gene that is a strong candidate for the male-determining factor. Data on humans and on laboratory and vole mice showing abnormalities of the sex determination mechanisms indicate that testis development depends on the presence of a testis-determining factor gene functioning in cooperation with X-linked and autosomal genes. Ovary development would depend on the absence of the testis-determining factor and perhaps on an alternative splicing of the transcripts from autosomal and X-linked genes involved in sex determination.

Genetic evidence equating SRY and the testis-determining factor

The testis-determining factor gene (TDF) lies on the Y chromosome and is responsible for initiating male sex determination. SRY is a gene located in the sex-determining region of the human and mouse Y chromosomes and has many of the properties expected for TDF. Sex reversal in XY females results from the failure of the testis determination or differentiation pathways. Some XY females, with gonadal dysgenesis, have lost the sex-determining region from the Y chromosome by terminal exchange between the sex chromosomes or by other deletions. If SRY is TDF, it would be predicted that some sex-reversed XY females, without Y chromosome deletions, will have suffered mutations in SRY. We have tested human XY females and normal XY males for alterations in SRY using the single-strand conformation polymorphism assay and subsequent DNA sequencing. A de novo mutation was found in the SRY gene of one XY female: this mutation was not present in the patient's normal father and brother. A second variant was found in the SRY gene of another XY female, but in this case the normal father shared the same alteration. The variant in the second case may be fortuitously associated with, or predisposing towards sex reversal; the de novo mutation associated with sex reversal provides compelling evidence that SRY is required for male sex determination.

DETECTION PF Y-SPECIFIC DNA SEQUENCES AND H-Y ANTIGEN EXPRESSION IN XY INDIVIDUALS WITH DISGENETIC GONADS DEVELOPMENT

We studied four patients with 46,XY karyotype and disgenetic gonads. Two of them (M.F., S.P.) are affected by XY gonadal dysgenesis (Swyer's syndrome) with eunuchoid habitus, female external genitalia and streak gonads. One (R.M.) had mixed gonadal dysgenesis (rudimentary testis on the R and a contralateral streak gonad) and one (M.R.) with perineal hypospadias, had disgenetic gonads (streak on the R. and rudimentary testis in the L, both with gonadoblastoma). All patients had Mullerian duct derivatives. Clinical, endocrine and serologic data, as well as DNA hybridization results using ZFY probe, Page et al. Cell. 51: 1091, 1987) are shown in the Table.The patients with XY gonadal dysgenesis have the Y-specific DNA sequences detected by ZFY probe, which correspond to a region encompassing the, so called, testis determining factor gene(TDF). These cases probably represent the autosomal recessive form of Swyer's syndrome (Two of them belonging to the H-Y negative form). The patient with mixed gonadal dysgenesis expresses H-Y antigen and has the ZFY sequence). Therefore, his condition is probably due to a delay in testicular organogenesis (as indicated by gonadal assymetry, with partial testicular differentiation on the R). The same situation could conceivably occur in the patient with bilateral gonadoblastoma. Altogether, our data suggest the involvement of other loci, besides TDF, in the primary determination of sex in man.This work was sponsored partially by a grant from FINEP-PADCT n° 43,84.0804

XX TRUE HERMAPHRODITISM ASSOCIATED TO MULTIPLE MALFORMATION. ABSENCE OF Y-SPECIFIC DNA SEQUENCES AND NORMAL H-Y ANTIGEN EXPRESSION

True hermaphroditism occurs when ovarian and testicular tissues are present in the same patient and usually is not associated to other malformation. We studied a patient with ambiguous genitalia from day 6 of life, phallus measured 3 cm, urethral and vaginal orifices were separate, slight posterior labial fusion and left(L) gonad palpable in the inguinal region, X̄ chromatin on buccal smear was positive 28% and karyotype in peripheral lymphocytes 46.XX. hCG stimulation resulted in great testosterone increase (<10 to 832 ng/dl). Ultrasonography revealed uterus and tubes but no intrabdominal gonads. The patient was assigned a female sex and at 16 month exploratory laparotomy revealed uterus, tubes, an ovary on the right(R) and an ovotestis on the 1, which was removed. The patient had multiple malformation; cranial assymetry, telecantum greater on the L, low nasal bridge, fat and cutaneous hypertrophy in lower limbs and gluteus and with keratosis, specially on the dorsum of R foot. On the feet: normal halux on L and with clinodactiTia on the R, 2nd. finger with bilateral macrodactilia and R Clinidactilia and 4th. finger with bilateral campodactilia and L macrodactilia. Growth was normal (Ht. 73.6 cm at ly. 1mo.). The parents were young and not consanguineous and denied affected relatives. The mother was medicated, with piperidolate hydrochloride in the first trimester and ampicilin and cephalotin for urinary infection on the 4th, month of gestation, The patient was born full term with 3.400 g and 46 cm lenqth and had hypoglycemia. The patient had no Y-specific UNA sequences, as detected by ZFY probe in Southern hybridizations (the probe corresponds to a Y chromosome region encompassing the socalled testis determining factor gene, or TD(-). Expression of H-Y antigen assayed with monoclonal anti-H-Y antibody and an ELISA was normal. Although hermaphroditism and malformations could be unrelated, one can speculate that alteration of a single autosomal or X-linked locus could have caused the patients's condition. This work was sponsored partially by a grant from

Chromosome Mapping and Expression of a Putative Testis-Determining Gene in Mouse

Isolation and mapping of a mouse complementary DNA sequence (mouse Y-finger) encoding a multiple, potential zinc-binding, finger protein homologous to the candidate human testis-determining factor gene is reported. Four similar sequences were identified in Hind III-digested mouse genomic DNA. Two (7.2 and 2.0 kb) were mapped to the Y chromosome. Only the 2.0-kb fragment, however, was correlated with testis determination. Polymerase chain reaction analysis suggests both Y loci are transcribed in adult testes. A 3.6-kb fragment was mapped to the X chromosome between the T16H and T6R1 translocation breakpoints, and a fourth (6.0 kb) was mapped to chromosome 10. Hence, mYfin sequences have been duplicated several times in the mouse, although they are not duplicated in humans.

The sex-determining region of the human Y chromosome encodes a finger protein.

The presence or absence of the Y chromosome determines whether a mammalian embryo develops as a male or female. In humans, genetic deletion analysis of "sex-reversed" individuals has identified a small portion of the Y chromosome necessary and sufficient to induce testicular differentiation of the bipotential gonad. We report the cloning of a 230-kilobase segment of the human Y chromosome that contains some or all of the testis-determining factor gene (TDF), the master sex-determining locus. The cloned region spans the deletion in a female who carries all but 160 kilobases of the Y. Certain DNA sequences within this region were highly conserved during evolution; homologs occur on the Y chromosomes of all mammals examined. In particular, homologous sequences are found within the sex-determining region of the mouse Y chromosome. The nucleotide sequence of this conserved DNA on the human Y chromosome suggests that it encodes a protein with multiple "finger" domains, as first described in frog transcription factor IIIA. The encoded protein probably binds to nucleic acids in a sequence-specific manner, and may regulate transcription. Very similar DNA sequences occur on the X chromosome of humans and other mammals. We discuss the possibility that the Y-encoded finger protein is the testis-determining factor, and propose models of sex determination accommodating the finding of a related locus on the X chromosome. The presence of similar sequences in birds suggests a possible role not only in the XX/XY sex determination system of mammals, but also in the ZZ/ZW system of birds.

Exchange of terminal portions of X- and Y-chromosomal short arms in human XX males

In most human 'XX males', DNA sequences normally found on Yp, the short arm of the Y chromosome, are present on Xp, the short arm of the X chromosome. To establish whether this transfer involves a terminal portion of Yp, and whether a terminal portion of Xp is lost in the process, we followed the inheritance of pseudoautosomal restriction fragment length polymorphisms in two XX-male families. One XX male apparently inherited the entire pseudoautosomal region of his father's Y chromosome and no part of the pseudoautosomal region of his father's X chromosome. The second XX male also inherited the entire pseudoautosomal region of his father's Y, but in addition inherited a proximal portion of the pseudoautosomal region of his father's X. These findings argue that XX males result from the transfer of a terminal portion of Yp onto Xp in exchange for a terminal portion of Xp (ref. 7). This implies that the testis-determining factor gene (TDF) maps distally in the strictly sex-linked portion of Yp, near the pseudoautosomal domain. The XX males described here appear to result from single (and, at least in the second case, unequal) crossovers proximal to the pseudoautosomal region on Yp and proximal to or within the pseudoautosomal region on Xp.