Terje Svingen

Building the mammalian testis: origins, differentiation, and assembly of the component cell populations

Development of testes in the mammalian embryo requires the formation and assembly of several cell types that allow these organs to achieve their roles in male reproduction and endocrine regulation. Testis development is unusual in that several cell types such as Sertoli, Leydig, and spermatogonial cells arise from bipotential precursors present in the precursor tissue, the genital ridge. These cell types do not differentiate independently but depend on signals from Sertoli cells that differentiate under the influence of transcription factors SRY and SOX9. While these steps are becoming better understood, the origins and roles of many testicular cell types and structures-including peritubular myoid cells, the tunica albuginea, the arterial and venous blood vasculature, lymphatic vessels, macrophages, and nerve cells-have remained unclear. This review synthesizes current knowledge of how the architecture of the testis unfolds and highlights the questions that remain to be explored, thus providing a roadmap for future studies that may help illuminate the causes of XY disorders of sex development, infertility, and testicular cancers.

Hox transcription factors and their elusive mammalian gene targets

The Hox family of homeodomain transcription factors regulate numerous pathways during developmental and normal cellular processes. All Hox proteins recognise similar sequences in vitro yet display functional diversity in an in vivo environment. This review focuses on the transcriptional and functional specificity elicited by Hox proteins, giving an overview of homeodomain–DNA interactions and the gain of binding specificity through cooperative binding with cofactors. Furthermore, currently identified mammalian Hox target genes are presented, of which the most striking feature is that very few direct Hox targets have been identified. The direct targets participate in an array of cellular functions including organogenesis and cellular differentiation, cell adhesion and migration and cell cycle and apoptotic pathways. A further assessment of identified mammalian promoter targets and the contribution of bases outside the canonical recognition motif is given, highlighting roles they may play in either trans-activation or repression by Hox proteins.

Sox7 and Sox17 are strain-specific modifiers of the lymphangiogenic defects caused by Sox18 dysfunction in mice

Developmental defects caused by targeted gene inactivation in mice are commonly subject to strain-specific modifiers that modulate the severity of the phenotype. Although several genetic modifier loci have been mapped in mice, the gene(s) residing at these loci are mostly unidentified, and the molecular mechanisms of modifier action remain poorly understood. Mutations in Sox18 cause a variable phenotype in the human congenital syndrome hypotrichosis-lymphedema-telangiectasia, and the phenotype of Sox18-null mice varies from essentially normal to completely devoid of lymphatic vasculature and lethal, depending on the strain of the mice, suggesting a crucial role for strain-specific modifiers in this system. Here we show that two closely related Group F Sox factors, SOX7 and SOX17, are able to functionally substitute for SOX18 in vitro and in vivo. SOX7 and SOX17 are not normally expressed during lymphatic development, excluding a conventional redundancy mechanism. Instead, these genes are activated specifically in the absence of SOX18 function, and only in certain strains. Our studies identify Sox7 and Sox17 as modifiers of the Sox18 mutant phenotype, and reveal their mechanism of action as a novel mode of strain-specific compensatory upregulation.

SOX9 Regulates MicroRNA miR-202-5p/3p Expression During Mouse Testis Differentiation

ABSTRACT MicroRNAs are important regulators of developmental gene expression, but their contribution to fetal gonad development is not well understood. We have identified the evolutionarily conserved gonadal microRNAs miR-202-5p and miR-202-3p as having a potential role in regulating mouse embryonic gonad differentiation. These microRNAs are expressed in a sexually dimorphic pattern as the primordial XY gonad differentiates into a testis, with strong expression in Sertoli cells. In vivo, ectopic expression of pri-miR-202 in XX gonads did not result in molecular changes to the ovarian determination pathway. Expression of the primary transcript of miR-202-5p/3p remained low in XY gonads in a conditional Sox9-null mouse model, suggesting that pri-miR-202 transcription is downstream of SOX9, a transcription factor that is both necessary and sufficient for male sex determination. We identified the pri-miR-202 promoter that is sufficient to drive expression in XY but not XX fetal gonads ex vivo. Mutation of SOX9 and SF1 binding sites reduced ex vivo transactivation of the pri-miR-202 promoter, demonstrating that pri-miR-202 may be a direct transcriptional target of SOX9/SF1 during testis differentiation. Our findings indicate that expression of the conserved gonad microRNA, miR-202-5p/3p, is downstream of the testis-determining factor SOX9, suggesting an early role in testis development.

Loss of Wnt5a Disrupts Primordial Germ Cell Migration and Male Sexual Development in Mice

ABSTRACT Disruptions in the regulatory pathways controlling sex determination and differentiation can cause disorders of sex development, often compromising reproductive function. Although extensive efforts have been channeled into elucidating the regulatory mechanisms controlling the many aspects of sexual differentiation, the majority of disorders of sex development phenotypes are still unexplained at the molecular level. In this study, we have analyzed the potential involvement of Wnt5a in sexual development and show in mice that Wnt5a is male-specifically upregulated within testicular interstitial cells at the onset of gonad differentiation. Homozygous deletion of Wnt5a affected sexual development in male mice, causing testicular hypoplasia and bilateral cryptorchidism despite the Leydig cells producing factors such as Hsd3b1 and Insl3. Additionally, Wnt5a-null embryos of both sexes showed a significant reduction in gonadal germ cell numbers, which was caused by aberrant primordial germ cell migration along the hindgut endoderm prior to gonadal colonization. Our results indicate multiple roles for Wnt5a during mammalian reproductive development and help to clarify further the etiology of Robinow syndrome (OMIM 268310), a disease previously linked to the WNT5A pathway.

Antagonistic regulation of Cyp26b1 by transcription factors SOX9/SF1 and FOXL2 during gonadal development in mice

Sex determination in fetal germ cells depends on a balance between exposure to retinoic acid (RA) and the degradation of RA achieved by the testis‐specific expression of the catabolic cytochrome P450 enzyme, CYP26B1. Therefore, identification of factors regulating the expression of the Cyp26b1 gene is an important goal in reproductive biology. We used in situ hybridization to demonstrate that Cyp26b1 and transcription factor genes steroidogenic factor‐1 (Sf1) and Sry‐related HMG box 9 (Sox9) are coexpressed in Sertoli cells, whereas Cyp26b1 and Sf1 are coexpressed in Leydig cells in mouse fetal testes. In the mouse gonadal somatic cell line TM3, transfection of constructs expressing SOX9 and SF1 activated Cyp26b1 expression, independently of the positive regulator RA. In embryonic gonads deficient in SOX9 or SF1, Cyp26b1 expression was decreased relative to wild‐type (WT) controls, as measured by quantitative RT‐PCR (qRT‐PCR). Furthermore, qRT‐PCR showed that Cyp26b1 up‐regulation by SOX9/SF1 was attenuated by the ovarian transcription factor Forkhead box L2 (FOXL2) in TM3 cells, whereas in Foxl2‐null mice, Cyp26b1 expression in XX gonads was increased ~20‐fold relative to WT controls. These data support the hypothesis that SOX9 and SF1 ensure the male fate of germ cells by up‐regulating Cyp26b1 and that FOXL2 acts to antagonize Cyp26b1 expression in ovaries.—Kashimada, K., Svingen, T., Feng, C.‐W., Pelosi, E., Bagheri‐Fam, S., Harley, V. R., Schlessinger, D., Bowles, J., Koopman, P. Antagonistic regulation of Cyp26b1 by transcription factors SOX9/SF1 and FOXL2 during gonadal development in mice. FASEB J. 25, 3561–3569 (2011). www.fasebj.org

Identification of Suitable Normalizing Genes for Quantitative Real-Time RT-PCR Analysis of Gene Expression in Fetal Mouse Gonads

In biological research, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assays are commonly employed to study mRNA abundance in cells and tissues. This type of assay usually relies on assessing transcript abundance relative to constitutively expressed endogenous reference genes. Therefore, it is important that the reference genes themselves are stably expressed in the cells or tissues analyzed, independent of factors such as age, sex, disease or experimental manipulations. Since no gene is expressed at the same level in all cells at all times, suitable reference genes must be identified for the specific cellular system or tissue being investigated. Here, we sought to identify stably expressed endogenous reference genes during embryonic gonad development in the mouse. We measured the transcript abundance of 10 frequently employed normalizing genes, of which 4 were stably expressed in fetal gonads from 11.5 to 14.5 dpc irrespective of sex. Based on our analysis, we suggest that Rn18s, Rps29, Tbp and Sdha are suitable reference genes for qRT-PCR expression studies during early gonad differentiation in the mouse.

Defective survival of proliferating Sertoli cells and androgen receptor function in a mouse model of the ATR-X syndrome

X-linked ATR-X (alpha thalassemia, mental retardation, X-linked) syndrome in males is characterized by mental retardation, facial dysmorphism, alpha thalassemia and urogenital abnormalities, including small testes. It is unclear how mutations in the chromatin-remodeling protein ATRX cause these highly specific clinical features, since ATRX is widely expressed during organ development. To investigate the mechanisms underlying the testicular defects observed in ATR-X syndrome, we generated ScAtrxKO (Sertoli cell Atrx knockout) mice with Atrx specifically inactivated in the supporting cell lineage (Sertoli cells) of the mouse testis. ScAtrxKO mice developed small testes and discontinuous tubules, due to prolonged G2/M phase and apoptosis of proliferating Sertoli cells during fetal life. Apoptosis might be a consequence of the cell cycle defect. We also found that the onset of spermatogenesis was delayed in postnatal mice, with a range of spermatogenesis defects evident in adult ScAtrxKO mice. ATRX and the androgen receptor (AR) physically interact in the testis and in the Sertoli cell line TM4 and co-operatively activate the promoter of Rhox5, an important direct AR target. We also demonstrate that ATRX directly binds to the Rhox5 promoter in TM4 cells. Finally, gene expression of Rhox5 and of another AR-dependent gene, Spinlw1, was reduced in ScAtrxKO testes. These data suggest that ATRX can directly enhance the expression of androgen-dependent genes through physical interaction with AR. Recruitment of ATRX by DNA sequence-specific transcription factors could be a general mechanism by which ATRX achieves tissue-specific transcriptional regulation which could explain the highly specific clinical features of ATR-X syndrome when ATRX is mutated.

Altered HOX Gene Expression in Human Skin and Breast Cancer Cells

Human HOX genes are expressed in a spatio-temporal fashion during embryogenesis and early development where they act as master transcriptional regulators. HOX genes are also expressed in normal adult cells, potentially in a tissue specific manner involving maintenance of the normal phenotype. In selected oncogenic transformations, mis-expression of many HOX genes have been shown, indicating an involvement of these transcriptional regulators in carcinogenesis and metastasis. Utilising quantitative real-time RT-PCR assays, the expression of 20 HOX genes and two known HOX co-factors, PBX1B and MEIS1, were analysed in human melanoma and breast cancer cell lines, comparing results against non-malignant cells. Alterations in HOX gene expression were observed for all malignant cells examined, with some dysregulated transcript levels seemingly random, and the expression of other HOX genes apparently following the same patterns in both skin and breast cancer establishment. Furthermore, HOX gene expression was correlated with the invasive capacity of the cells. The expression of the HOX co-factors PBX1B and MEIS1 showed no marked changes from the non-malignant to the malignant phenotypes, further indicating that it is dysregulated HOX gene expression, rather than dysregulated gene expression of HOX co-factors, that potentially commit the cell to re-differentiate and undergo oncogenic transformation.

Female-to-male sex reversal in mice caused by transgenic overexpression of Dmrt1.

Genes related to Dmrt1, which encodes a DNA-binding DM domain transcription factor, act as triggers for primary sex determination in a broad range of metazoan species. However, this role is fulfilled in mammals by Sry, a newly evolved gene on the Y chromosome, such that Dmrt1 has become dispensable for primary sex determination and instead maintains Sertoli cell phenotype in postnatal testes. Here, we report that enforced expression of Dmrt1 in XX mouse fetal gonads using a Wt1-BAC transgene system is sufficient to drive testicular differentiation and male secondary sex development. XX transgenic fetal gonads showed typical testicular size and vasculature. Key ovarian markers, including Wnt4 and Foxl2, were repressed. Sertoli cells expressing the hallmark testis-determining gene Sox9 were formed, although they did not assemble into normal testis cords. Other bipotential lineages differentiated into testicular cell types, including steroidogenic fetal Leydig cells and non-meiotic germ cells. As a consequence, male internal and external reproductive organs developed postnatally, with an absence of female reproductive tissues. These results reveal that Dmrt1 has retained its ability to act as the primary testis-determining trigger in mammals, even though this function is no longer normally required. Thus, Dmrt1 provides a common thread in the evolution of sex determination mechanisms in metazoans. Highlighted article: Even though its function is not normally required for sex determination in mammals, Dmrt1 is able to drive sex reversal in XX mice, suggesting that it has retained sex-determining function.