XY Testes Research Articles

Nr5a1 suppression during the murine fetal period optimizes ovarian development by fine-tuning Notch signaling

The nuclear receptor NR5A1 is equally expressed and required for development of the gonadal primordia of both sexes, but, after sex determination, it is upregulated in XY testes and downregulated in XX ovaries. We have recently demonstrated, in mice, that this downregulation is mediated by forkhead box L2 (FOXL2) and hypothesized that adequate suppression of Nr5a1 is essential for normal ovarian development. Further, analysis of human patients with disorders/differences of sex development suggests that overexpression of NR5A1 can result in XX (ovo)testicular development. Here, we tested the role of Nr5a1 by overexpression in fetal gonads using a Wt1-BAC (bacterial artificial chromosome) transgene system. Enforced Nr5a1 expression compromised ovarian development in 46,XX mice, resulting in late-onset infertility, but did not induce (ovo)testis differentiation. The phenotype was similar to that of XX mice lacking Notch signaling. The expression level of Notch2 was significantly reduced in Nr5a1 transgenic mice, and the ovarian phenotype was almost completely rescued by in utero treatment with a NOTCH2 agonist. We conclude that suppression of Nr5a1 during the fetal period optimizes ovarian development by fine-tuning Notch signaling.

Comparative Transcriptome Analysis of Differentially Expressed Genes and Signaling Pathways between XY and YY Testis in Yellow Catfish.

YY super-males have rarely been detected in nature and only been artificially created in some fish species including tilapia and yellow catfish (Pelteobagrusfulvidraco), which provides a promising model for testis development and spermatogenesis. In our previous study, significant differences in morphology and miRNA expression were detected between XY and YY testis of yellow catfish. Here, solexa sequencing technology was further performed to compare mRNA expression between XY and YY testis. Compared with unigenes expressed in XY testis, 1146 and 1235 unigenes have significantly higher and lower expression in YY testis, respectively. 605 differentially expressed unigenes were annotated to 1604 GO terms with 319 and 286 genes having relative higher expression in XY and YY testis. KEGG analysis suggested different levels of PI3K-AKT and G protein-coupled receptor (GPCR) signaling pathways between XY and YY testis. Down-regulation of miR-141/429 in YY testis was speculated to promote testis development and maturation, and several factors in PI3K-AKT and GPCR signaling pathways were found as predicted targets of miR-141/429, several of which were confirmed by dual-luciferase reporter assays. Our study provides a comparative transcriptome analysis between XY and YY testis, and reveals interactions between miRNAs and their target genes that are possibly involved in regulating testis development and spermatogenesis.

Sex-biased miRNAs in gonad and their potential roles for testis development in yellow catfish.

Recently, YY super-male yellow catfish had been created by hormonal-induced sex reversal and sex-linked markers, which provides a promising research model for fish sex differentiation and gonad development, especially for testis development. MicroRNAs (miRNAs) have been revealed to play crucial roles in the gene regulation and gonad development in vertebrates. In this study, three small RNA libraries constructed from gonad tissues of XX female, XY male and YY super-male yellow catfish were sequenced. The sequencing data generated a total of 384 conserved miRNAs and 113 potential novel miRNAs, among which 23, 30 and 14 miRNAs were specifically detected in XX ovary, XY testis, and YY testis, respectively. We observed relative lower expression of several miR-200 family members, including miR-141 and miR-429 in YY testis compared with XY testis. Histological analysis indicated a higher degree of testis maturity in YY super-males compared with XY males, as shown by larger spermatogenic cyst, more spermatids and fewer spermatocytes in the spermatogenic cyst. Moreover, five miR-200 family members were significantly up-regulated in testis when treated by 17α-ethinylestradiol (EE2), high dose of which will impair testis development and cell proliferation. The down-regulation of miR-141 and 429 coincides with the progression of testis development in both yellow catfish and human. At last, the expression pattern of nine arbitrarily selected miRNAs detected by quantitative RT-PCR was consistent with the Solexa sequencing results. Our study provides a comprehensive miRNA transcriptome analysis for gonad of yellow catfish with different sex genotypes, and identifies a number of sex-biased miRNAs, some of that are potentially involved in testis development and spermatogenesis.

Gene Expression Profile During Testicular Development in Patients With SRY-negative 46,XX Testicular Disorder of Sex Development

To elucidate alternative pathways in testicular development, we attempted to clarify the genetic characteristics of SRY-negative XX testes. We previously reported 5 cases of SRY-negative 46,XX testicular disorders of sex development and demonstrated that coordinated expression of genes such as SOX9, SOX3, and DAX1 was associated with testicular development. We performed a case-control study between the aforementioned boy with 46,XX testicular disorders of sex development and an age-matched patient with hydrocele testis (46,XY). During their consecutive surgeries, testicular biopsy specimens were obtained. Genes with differential expression compared with XY testis were identified using polymerase chain reaction (PCR)-based subtractive hybridization and sequencing. For validation of differential gene expression, real-time RT-PCR was performed using gene-specific primers. The distribution of candidate proteins in the testicular tissue was clarified by immunohistochemistry in human and rodent specimens. Moreover, in vitro inhibitory assays were performed. We identified 13 upregulated and 7 downregulated genes in XX testis. Among the candidate genes, we focused on ROCK1 (Rho-associated, coiled-coil protein kinase 1) in the upregulated gene group, because high expression in XX testis was validated by real-time RT-PCR. ROCK1 protein was detected in germ cells, Leydig cells, and Sertoli cells by immunohistochemistry. Moreover, the addition of specific ROCK1 inhibitor to Sertoli cells decreased SOX9 gene expression. On the basis of in vitro inhibitory assay, it is suggested that ROCK1 phosphorylates and activates SOX9 in Sertoli cells. Testes formation might be initiated by an alternative signaling pathway attributed to ROCK1, not SRY, activation in XX testes.

813 FUNCTIONAL ANALYSIS OF RHO SIGNALING CASCADE CONCERNED WITH CELL ADHESION IN THE PROCESS OF TESTICULAR DEVELOPMENT

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research (I)1 Apr 2013813 FUNCTIONAL ANALYSIS OF RHO SIGNALING CASCADE CONCERNED WITH CELL ADHESION IN THE PROCESS OF TESTICULAR DEVELOPMENT Kentaro Mizuno, Yutaro Hayashi, Hideyuki Kamisawa, Yoshinobu Moritoki, Hidenori Nishio, Makoto Imura, Yasuhiro Shibata, Satoshi Kurokawa, Akihiro Nakane, Toshiki Kato, Tetsuji Maruyama, Yoshiyuki Kojima, and Kenjiro Kohri Kentaro MizunoKentaro Mizuno Nagoya, Japan More articles by this author , Yutaro HayashiYutaro Hayashi Nagoya, Japan More articles by this author , Hideyuki KamisawaHideyuki Kamisawa Nagoya, Japan More articles by this author , Yoshinobu MoritokiYoshinobu Moritoki Nagoya, Japan More articles by this author , Hidenori NishioHidenori Nishio Nagoya, Japan More articles by this author , Makoto ImuraMakoto Imura Nagoya, Japan More articles by this author , Yasuhiro ShibataYasuhiro Shibata Nagoya, Japan More articles by this author , Satoshi KurokawaSatoshi Kurokawa Nagoya, Japan More articles by this author , Akihiro NakaneAkihiro Nakane Nagoya, Japan More articles by this author , Toshiki KatoToshiki Kato Nagoya, Japan More articles by this author , Tetsuji MaruyamaTetsuji Maruyama Nagoya, Japan More articles by this author , Yoshiyuki KojimaYoshiyuki Kojima Fukushima, Japan More articles by this author , and Kenjiro KohriKenjiro Kohri Nagoya, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.379AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES During early stage of testis development, formation of seminiferous tubule is preceded with adhesion between germ cells and somatic cells. However, the relationship between the mechanisms of testis development and cell adhesion remains unclear. Previously, we detected Rock1 gene as testis development related gene, and Rock1 gene is belong to Rho signaling cascade which regulate cell adhesion. In this study, to clarify the role of cell adhesion in the process of testis development comprehensively, we performed functional analysis of factors concerned with Rho signaling cascade in testis. METHODS From testicular tissue samples of patients with 46,XX testicular DSD (XX testis) and age-matched patients with hydrocele testis (XY testis), mRNA was extracted and cDNA libraries were obtained, respectively. We explored the genes associated with differential expression between XY and XX testis using by PCR-subtractive hybridization methods. Subsequently, we detected that ROCK1 gene is up-regulated in XX testis without SRY gene. We investigated distribution of not only Rock1 protein but also β1-integrin and Rho proteins which are upstream of Rho signaling cascade in testicular tissues of human and experimental animals. Furthermore, to analyze the function of Rock1 gene, Y-27632, selective inhibitor of Rock1, was added into cultured Sertoli cells and expression changes of each gene including Rho signaling cascade were examined by RT-PCR. This study was approved by the institutional review board in our hospital. RESULTS ROCK1 protein was detected in spermatogonia and Sertoli cells in immature testis of human. While, in adult mice, Rock1 protein was detected in spermatocytes, spermatids and Leydig cells in seminiferous tubule stage-specific manner. β1-integrin protein was detected in germ cells as well as Rock1 protein. In addition of Y-27632 into cultured Sertoli cells, expressions of Sox9 gene were significantly decreased, but expression changes of RhoA and RhoB gene, which is upstream of Rock1 gene, were not significant. CONCLUSIONS Rock1 protein was expressed in both germ cells and somatic cells, suggesting that Rock1 is associated with cell adhesion in each other. Furthermore, we elucidated that Sox9 gene is up-regulated by Rock1 gene and regulated by several signaling pathway. Since it is reported that niche surrounding spermatogonial stem cells is impaired in knockout mice of Sox9 gene, it is suggested that Rho signaling cascade upregulates Sox9 gene and is concerned with testis development through adhesion between germ cells and somatic cells. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e334 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kentaro Mizuno Nagoya, Japan More articles by this author Yutaro Hayashi Nagoya, Japan More articles by this author Hideyuki Kamisawa Nagoya, Japan More articles by this author Yoshinobu Moritoki Nagoya, Japan More articles by this author Hidenori Nishio Nagoya, Japan More articles by this author Makoto Imura Nagoya, Japan More articles by this author Yasuhiro Shibata Nagoya, Japan More articles by this author Satoshi Kurokawa Nagoya, Japan More articles by this author Akihiro Nakane Nagoya, Japan More articles by this author Toshiki Kato Nagoya, Japan More articles by this author Tetsuji Maruyama Nagoya, Japan More articles by this author Yoshiyuki Kojima Fukushima, Japan More articles by this author Kenjiro Kohri Nagoya, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Involvement of FGF9/16/20 subfamily in female germ cell development of the Nile tilapia, Oreochromis niloticus

Fibroblast growth factors (FGFs) have been proved to participate in a wide variety of processes, including growth, differentiation, cell proliferation, migration, sex determination and sex differentiation. The roles of FGF9/16/20 subfamily members in the gonadal development of teleost fish have not yet been reported. Three FGFs (16, 20a and 20b) of the FGF9/16/20 subfamily were cloned from the Nile tilapia by RT-PCR and RACE. Phylogenetic, bioinformatic and syntenic analyses demonstrated that these cloned FGFs are genuine FGF16, 20a and 20b. Our analyses further supported the non-existence of FGF9 ortholog and the existence of two FGF20 paralogs in teleost genomes. Tissue distribution analysis by RT-PCR demonstrated that FGF16 was expressed in a wide range of tissues including the testis and ovary, FGF20b in the brain, pituitary, intestine and ovary, but not in the testis, while FGF20a in the brain, pituitary and spleen, but not in the gonad. These results were consistent with the Northern blot analysis. The expression profiles of FGF16 and FGF20b during normal and sex reversed gonadal development were investigated by real-time PCR. Both showed much higher expression in the XX ovary and 17 beta-estradiol induced XY ovary compared with the XY testis and fadrozole and tamoxifen induced XX testis, with the highest in both sexes at 120dah. Strong signals of FGF16 and FGF20b were detected in phase II oocytes by in situ hybridization. These data suggest that FGF9/16/20 subfamily is involved in the early oocyte development of the female.

Antagonism of the testis- and ovary-determining pathways during ovotestis development in mice

Ovotestis development in B6-XY POS 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-XY POS 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.

Up‐regulation of SOX9 in human sex‐determining region on the Y chromosome (SRY)‐negative XX males

In mammals, gonadal sex is normally determined by the presence or absence of the Y chromosome gene SRY. After expression of SRY in the sexually indifferent gonad, a number of genes encoding transcription factors and growth factors implicated in testis differentiation start to show male-specific expression. However, in XX males, these genes must be up-regulated in the absence of SRY, but the aetiology of SRY-negative XX maleness remains unclear. We examined the expression of representative gonad marker genes in SRY-negative XX male testes. RT-PCR and immunohistochemical studies revealed that SOX9, DAX-1, Ad4BP/SF-1, WT-1, GATA-4 and MIS were expressed in testicular tissues of SRY-negative XX males. Expression levels of SOX9 in testes of these patients averaged 1.9-fold higher than in normal XY testes, while expression levels of Ad4BP/SF-1, DAX-1 and MIS were lower in the SRY-negative XX testes than in XY testes. All XX patients were found to carry two copies of the SOX9 gene per diploid genome as do normal XX females and XY males. The XX male patients also carried two copies of the DAX-1 gene as do normal XX females, while normal XY males carry a single DAX-1 gene. Our data suggest that lesions affecting SOX9 expression are the key factor in sex determination in SRY-negative XX males, and that the decreased expression of Ad4BP/SF-1, DAX-1 and MIS contribute to their clinical features.

Two DM domain genes, DMY and DMRT1, involved in testicular differentiation and development in the medaka, Oryzias latipes

The recent discovery of the DMY gene (DM domain gene on Y chromosome and one of the DMRT1 family genes) as a key determinant of male development in the medaka (Oryzias latipes) has led to its designation as the prime candidate gene for sex-determination in this species. This study focused on the sites and pattern of expression of DMY and DMRT1 genes during gonadal differentiation of medaka to further determine their roles in testis development. DMY mRNA and protein are expressed specifically in the somatic cells surrounding primordial germ cells (PGCs) in the early gonadal primordium, before morphological sex differences are seen. However, somatic cells surrounding PGCs never express DMY during the early migratory period. Expression of DMY persists in Sertoli cell lineage cells, from PGC-supporting cells to Sertoli cells, indicating that only DMY-positive cells enclose PGCs during mitotic arrest after hatching. DMRT1 is expressed in spermatogonium-supporting cells after testicular differentiation (20-30 days after hatching), and its expression is much higher than that of DMY in mature testes. In XX sex-reversed testes, DMRT1 is expressed in the Sertoli cell lineage, similar to the expression of DMY in XY testes. These results suggest strongly that DMY regulates PGC proliferation and differentiation sex-specifically during early gonadal differentiation of XY individuals and that DMRT1 regulates spermatogonial differentiation.

Biopsied testis cells of four 47,XXY patients: fluorescence in-situ hybridization and ICSI results.

A testis biopsy was performed for four non-mosaic 47,XXY azoospermic patients. Spermatozoa were found in three cases and frozen before ICSI. We analysed the various cells found in the four samples by multicolour fluorescence in-situ hybridization (FISH), to evaluate the meiosis and spermatogenesis possibilities of the 47,XXY and 46,XY testis cell lines, and to estimate aneuploidy rate in the resulting spermatids and spermatozoa. Testis diploid cells (either somatic or premeiotic), meiotic, and post-meiotic haploid germ cells were hybridized with probes for chromosomes X, Y and 18. The only patient with no spermatozoa had a homogeneous diploid XXY constitution in the testis; the three other patients presented two cell populations (46,XY and 47,XXY) among their diploid testis cells. All the observed pachytene figures were XY; no XXY pachytene figure was found. The aneuploidy rate among post-meiotic cells for chromosomes X,Y and 18 was 6.75% (5/74). This rate was 1.5% (2/133) for control. Three couples underwent ICSI; four attempts were made, one healthy baby was born. FISH results suggest that only 46,XY cells can undergo meiosis.

Sex chimaerism, fertility and sex determination in the mouse

Adult intraspecific mouse chimaeras, derived by introducing male embryonal stem cells into unsexed host blastocysts, were examined to determine whether gonadal sex was correlated with the sex chromosome composition of particular cell lineages. The fertility of XX in equilibrium XY and XY in equilibrium XY male chimaeras was also compared. The distribution of XX and XY cells in 34 XX in equilibrium XY ovaries, testes and ovotestes was determined by in situ hybridisation using a Y-chromosome-specific probe. Both XX and XY cells were found in all gonadal somatic tissues but Sertoli cells were predominantly XY and granulosa cells predominantly XX. The sex chromosome composition of the tunica albuginea and testicular surface epithelium could not, in general, be fully resolved, owing to diminished hybridisation efficiency in these tissues, but the ovarian surface epithelium (which like the testicular surface epithelium derives from the coelomic epithelium) was predominantly XX. These findings show that the claim that Sertoli cells were exclusively XY, on which some previous models of gonadal sex determination were based, was incorrect, and indicate instead that in the mechanism of Sertoli cell determination there is a step in which XX cells can be recruited. However, it remains to be established whether the sex chromosome constitution of the coelomic epithelium lineage plays a causal role in gonadal sex determination. Male chimaeras with XX in equilibrium XY testes were either sterile or less fertile than chimaeras with testes composed entirely of XY cells. This impaired fertility was associated with the loss of XY germ cells in atrophic seminiferous tubules. Since this progressive lesion was correlated with a high proportion of XX Leydig cells, we suggest that XX Leydig cells are functionally defective, and unable to support spermatogenesis.

In situ analysis of fetal, prepuberal and adult XX ↔ XY chimaeric mouse testes: Sertoli cells are predominantly, but not exclusively, XY

The testes of fetal, prepuberal and adult XX----XY chimaeras were examined using in situ hybridisation to identify the beta-globin transgenic marker contained in one component of each chimaera. This enabled the proportion of XX and XY cells contributing to the major cell lineages of the testis to be estimated from sectioned and air-dried material. A few XX Sertoli cells were found in all three age groups, but the XX contribution was always much lower than in other somatic cell types. Significantly, in fetal XX----XY testes, Sertoli cells were the only cell type to show a bias in favour of the XY component. This strengthens the view that Tdy acts solely in the lineage that gives rise to Sertoli cells. However, the finding of some fetal XX Sertoli cells means that one of the steps in the Tdy-initiated process of Sertoli cell determination is capable of locally recruiting XX cells.

Detailed structure of the Drosophila melanogaster stellate genes and their transcripts.

The X-linked Stellate locus contains two major size classes of a tandemly repeated gene. An example of each class has been sequenced. The steady-state level of Stellate RNA is much higher in XO testis than in XY testis. Sequencing of six cDNA clones derived from XO testis RNA shows that there are two major introns in the Stellate genes. Primer extension and RNase protection analyses show that these introns are spliced much more efficiently in XO than in XY testis. These results also indicate the major transcriptional start site for Stellate RNA. P element transformation results with a marked Stellate gene demonstrate that at least one of the genes sequenced contains a functional promoter, which generates low levels of RNA in XY testis and high levels of RNA in XO testis. This promoter does not contain a TATA element in the -30 region relative to the transcriptional start. Previous results had implicated a specific region of the Y chromosome, designated here as the Su(Ste) locus, in the control of the Stellate genes on the X. Analysis using segmental Y deficiencies shows that the Su(Ste) region suppresses both the high levels and efficient splicing of Stellate RNA.

A mouse zinc finger gene which is transiently expressed during spermatogenesis.

Zinc finger proteins are polypeptides with sequence-specific, nucleic acid-binding properties. Substantial evidence has established them as a class of trans-acting molecules with regulatory roles in cellular growth and differentiation. We have screened an 11.5 day post coitum urogenital ridge cDNA library with an oligonucleotide encoding a sequence conserved between a variety of zinc finger proteins. By cDNA cloning and sequencing we show that a novel mouse gene, Zfp-35, encodes a protein with a block of 18 zinc finger domains and an N-terminal region rich in acidic residues. The 2.4 kb mRNA encoding this polypeptide is selectively expressed in adult testis, by comparison with other organs. We have analysed Zfp-35 expression in whole testes of sex-reversed mice, whole testes of prepuberal XY animals, germ cell fractions from XY adult testes and by in situ hybridization to sections from adult XY testes. Our studies show that a considerable increase in expression is restricted to spermatocytes at the pachytene stage of meiotic prophase. These experiments suggest that Zfp-35 may act to control gene activity during this particular stage of spermatogenesis.

Identical action of Y chromosome and sxr factor in early testicular development in the mouse.

The Sxr (sex reversed) factor in the mouse described by Cattanach et al. (1971) causes sex reversal of genetic female mice, thus producing XX males. Its primary effect is induction of testicular development in XX embryos. In the present study, the early steps of testicular development in sex reversed XX and in XY mouse embryos are examined. The embryos are bred from male carriers of the Sxr mutation. After determination of the sex chromosomes from chromosomal spreads, the appearance of testis cords is evaluated in sex reversed XX embryos and their XY littermates at 12 1/2 and 13 days of pregnancy. In sex reversed XX embryos testis cords appear at the same stage of development as in XY embryos. From day 13 to 17 of pregnancy, the volume of the testis anlage is determined by planimetry in sex reversed XX and in normal XY embryos from separate litters. No significant difference is found in the growth rate of XX and XY testes. Measurements of the diameter of the testis cords also indicate the same developmental pattern in both groups. Thus, during induction of a testis the Sxr factor perfectly imitates the Y chromosome in all parameters investigated.