molecular development

summary

Development of the gonads (testes or ovaries) requires the coordinated functioning of many genes and proteins. A breakdown at any point in this complex developmental pathway may lead to a child of uncertain sex.

Children with intersex disorders occur one in every 4,500 births and are a major paediatric issue.

Our research focuses on sex determination, and aims to understand how genes control gonad development. This knowledge will improve the care of intersex children, and will provide insights into diseases that affect this and other organ systems.

group leader(s)

  Professor Andrew Sinclair
  Molecular Development
  Murdoch Childrens Research Institute
  Flemington Road
  Parkville Victoria 3052

  T   +61 3 8341 6425
  F   +61 3 8341 6429
  E   andrew.sinclair@mcri.edu.au

group leader biography

current research projects

Project 1: Intersex patient analysis using high density Single Nucleotide Polymorphism (SNP) Microarrays

Intersex disorders, ranging in severity from genital abnormalities to complete sex reversal, are surprisingly common. Uncertainty about a child's gender is extremely traumatic for the individual, parents and other family members. There are profound psychological and reproductive consequences in later life for the affected patient. The cause of these problems is most often the failure of the complex network of genes that regulate development of testes or ovaries. Our research seeks to understand the molecular and cellular basis of testis and ovary development and how mutations in key genes can lead to abnormalities. We have identified several major genes that play critical roles in testis development. Mutations in these genes explain 20% of intersex cases but we have no explanation for the remaining 80% of patients. In order to find these other genes that affect gonad development we will use the Affymetrix human gene chip comprised of 500,000 single nucleotide polymorphism (SNP) markers covering the entire human genome. DNA from intersex patients will be used to interrogate these chips for micro-deletions and micro-duplications in the human genome. Data from the chips will be subjected to detailed bioinformatic analysis to pinpoint novel genes responsible for gonad development. These genes will be subsequently analysed in mouse models to determine their precise molecular/ cellular function in testis and ovary development.

Project 2: The molecular genetics of gonad development and dysfunction

We have used a variety of screens in mouse to isolate genes that are differentially expressed from either the male or female developing gonad. Such differential expression suggests a potential role in either testis or ovary development. We are currently conducting a range of functional assays on these genes in mouse embryos to determine the role of these genes in developing gonads. Null mice have been created for several genes and these are being examined in detail for phenotypic changes in gonad development. However, many other genes are being analysed using more rapid assays. These rapid approaches include siRNA knockdown and over-expression in embryonic mouse gonad cultures. The effects of knockdown or over-expression are being monitored by changes in histology of the gonad and by assessing perturbations in the expression profile of known genes using real-time quantitative PCR. These more rapid functional assays will help assign a number of genes to the testis or ovary determining regulatory network. This work is closely integrated with Project 1 as many of the mouse models of gonad development will find correlates in human intersex patients.

Project 3: Analysis of genes responsible for infertility and germ cell cancers

Germ cells are unique in that they contribute to the next generation. To reiterate the program of embryonic development germ cells must retain underlying information that will allow them to generate new cells or 'totipotency'. In this regard germ cells appear similar to embryonic stem (ES) cells. Using molecular and computer based screens we have identified genes expressed in embryonic stem cells and during embryonic germ line development. These genes may function in establishing/maintaining totipotency in ES cells and early germ cells or may have other functions during later germ line development. This project uses in situ hybridisation and immunohistochemical techniques to examine the expression of candidate germ line genes during mouse embryonic development. Based on their expression pattern the genes likely have important germ line roles will be functionally examined in ES cells and developing embryos using a lentiviral gene delivery system to increase (over-expression), or reduce expression (using RNA interference). This work will address the function of these genes in the developing mouse germ line and their possible roles in human infertility and germ cell cancers.

team members

• Andrew Cutting - PhD Student (UoM Paeds)
• Stefanie Eggers - PhD Student (UoM Paeds)
• Katie Griffin - PERSONAL ASSISTANT
• Sonia Grover - HONORARY RESEARCH FELLOW
• Sonja Gustin - Research Assistant
• Jacky Hewitt - PhD Student (UoM Paeds)
• Thomas Ohnesorg - Postdoctoral Fellow
• Kelly Roeszler - Research Assistant
• Jacqueline Tan - Postdoc Scientist
• Jocelyn Van Den Bergen - Research Assistant

publications

• Bagheri-Fam S., Argentaro A., Svingen T., Combes AN., Sinclair AH., Koopman P., Harley VR. Defective survival of proliferating Sertoli cells and androgen receptor function in a mouse model of the ATR-X syndrome. HUMAN MOLECULAR GENETICS 20 (11) : 2213 - 2224(2011) PubMed
• Bannister SC., Smith CA., Roeszler KN., Doran TJ., Sinclair AH., Tizard MLV. Manipulation of Estrogen Synthesis Alters MIR202*Expression in Embryonic Chicken Gonads. BIOLOGY OF REPRODUCTION 85 (1) : 22 - 30(2011) PubMed
• Bernard P., Ryan J., Sim H., Czech D., Sinclair AH., Koopman P., Harley VR. Wnt Signaling in Ovarian Development Inhibits Sf1 Activation of Sox9 via the Tesco Enhancer. ENDOCRINOLOGY (2011)
• Cutting AD., Bannister SC., Doran TJ., Sinclair AH., Tizard MV., Smith CA. The Potential role of microRNAs in regulating gonadal sex differentiation in the chicken embryo. Chromosome research (2011)
• Gillam LH., Hewitt JK., Warne GL. Ethical principles: An essential part of the process in disorders of sex development care. Hormone Research in Paediatrics 76 (5) : 367 - 368(2011) PubMed
• Jayasinghe YL., Moore EE., Tabrizi SN., Grover SR., Garland SM. Human papillomavirus in adolescents: Lessons learned from decades of evaluation. JOURNAL OF PAEDIATRICS AND CHILD HEALTH (2011) PubMed
• Kimberley N., Hutson JM., Southwell BR., Grover SR. Vaginal agenesis, the hymen, and associated anomalies. Journal of Pediatric and Adolescent Gynecology (2011) PubMed
• Kimberley N., Hutson JM., Southwell BR., Grover SR. Well-being and sexual function outcomes in women with vaginal agenesis. FERTILITY AND STERILITY 95 (1) : 238 - 241(2011) PubMed
• Ohnesorg T., Turbitt E., White SJ. The many faces of MLPA. Methods in Molecular Biology 687 : 193 - 205(2011) PubMed
• Peake LJ., Grover SR., Monagle PT., Kennedy AD. Effect of warfarin on menstruation and menstrual management of the adolescent on warfarin. JOURNAL OF PAEDIATRICS AND CHILD HEALTH (2011) PubMed
• Ryan J., Ludbrook L., Wilhelm D., Sinclair A., Koopman P., Bernard P., Harley VR. Analysis of Gene Function in Cultured Embryonic Mouse Gonads Using Nucleofection. SEXUAL DEVELOPMENT 5 (1) : 7 - 15(2011) PubMed
• Sim H., Argentaro A., Czech DP., Bagheri-Fam S., Sinclair AH., Koopman P., Boizet-Bonhoure B., Poulat F., Harley VR. Inhibition of SRY-Calmodulin Complex Formation Induces Ectopic Expression of Ovarian Cell Markers in Developing XY Gonads. ENDOCRINOLOGY 152 (7) : 2883 - 2893(2011) PubMed
• Western PS., Ralli RA., Wakeling SI., Lo C., van den Bergen JA., Miles DC., Sinclair AH. Mitotic Arrest in Teratoma Susceptible Fetal Male Germ Cells. PLOS ONE 6 (6) (2011) PubMed
• White S., Hewitt J., Turbitt E., van der Zwan Y., Hersmus R., Drop S., Koopman P., Harley V., Cools M., Looijenga L., Sinclair A. A multi-exon deletion within WWOX is associated with a 46,XY disorder of sex development. EUROPEAN JOURNAL OF HUMAN GENETICS (2011) PubMed
• White S., Ohnesorg T., Notini A., Roeszler K., Hewitt J., Daggag H., Smith C., Turbitt E., Gustin S., van den Bergen J., Miles D., Western P., Arboleda V., Schumacher V., Gordon L., Bell K., Bengtsson H., Speed T., Hutson J., Warne G., Harley V., Koopman P., Vilain E., Sinclair A. Copy Number Variation in Patients with Disorders of Sex Development Due to 46,XY Gonadal Dysgenesis. PLOS ONE 6 (3) (2011) PubMed