Comparison of global gene expression profiles of microdissected human foetal Leydig cells with their normal and hyperplastic adult equivalents.
Do human adult Leydig cells (ALCs) within hyperplastic micronodules display characteristics of foetal LCs (FLCs)?



The gene expression profiles of FLCs and all ALC subgroups were clearly different, but there were no significant differences in expressed genes between the normally clustered and hyperplastic ALCs.



LCs are the primary androgen producing cells in males throughout development and appear in chronologically distinct populations; FLCs, neonatal LCs and ALCs. ALCs are responsible for progression through puberty and for maintenance of reproductive functions in adulthood. In patients with reproductive problems, such as infertility or testicular cancer, and especially in men with high gonadotrophin levels, LC function is often impaired, and LCs may cluster abnormally into hyperplastic micronodules (defined as clusters of >15 LCs in a cross-section).



A genome-wide microarray study of LCs microdissected from human foetal and adult tissue samples (n = 12). Additional tissue specimens (n = 15) were used for validation of the mRNA expression data at the protein level.



Frozen human tissue samples were used for the microarray study, including morphologically normal foetal (gestational week 10-11) testis samples, and adult testis specimens with normal LC distribution, LC micronodules or LC micronodules adjacent to hCG-producing testicular germ cell tumours. Transcriptome profiling was performed on Agilent whole human genome microarray 4 × 44 K chips. Microarray data pre-processing and statistical analysis were performed using the limma R/Bioconductor package in the R software, and differentially expressed genes were further analysed for gene set enrichment using the DAVID Bioinformatics software. Selected genes were studied at the protein level by immunohistochemistry.



The transcriptomes of FLCs and ALCs differed significantly from each other, whereas the profiles of the normally clustered and hyperplastic ALCs were similar despite morphological heterogeneity. The study revealed several genes not known previously to be expressed in LCs during early development, including sulfotransferase family 2A member 1 (SULT2A1), WNT1-inducible signalling pathway protein 2 (WISP2), hydroxyprostaglandin dehydrogenase (HPGD) and insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1), whose expression changes were validated at the protein level.



The transcriptomic data are deposited in ArrayExpress (accession code E-MTAB-5453).



The small number of biological replicates and the necessity of RNA amplification due to the scarcity of human tissues, especially foetal specimens, are the main limitations of the study. Heterogeneous subpopulations of LCs within micronodules were not discriminated during microdissection and might have affected the expression profiling. The study was constrained by the lack of availability of truly normal controls. Testis samples used as 'controls' displayed complete spermatogenesis and were from patients with germ cell neoplasia but with undetectable hCG and normal hormone levels.



The changes in LC morphology and function observed in patients with reproductive disorders possibly reflect subtle changes in the expression of many genes rather than regulatory changes of single genes or pathways. The study provides new insights into the development and maturation of human LCs by the identification of a number of potential functional markers for FLC and ALC.



The study was supported by research grants from the Danish Cancer Society, the Capital Region's Research Fund for Health Research, Rigshospitalet's research funds, the Villum Kann Rasmussen Foundation, the Danish Innovation Fund, ReproUnion, Kirsten and Freddy Johansen's foundation and the Novo Nordisk Foundation. None of the funding agencies had any influence on the study. The authors declare no conflicts of interest.
Department of Growth and Reproduction, Copenhagen University Hospital(Rigshospitalet), International Center for Research and Training in Endocrine Disruption of Male Reproduction & Child Health (EDMaRC), 9 Blegdamsvej, DK-2100 Copenhagen, Denmark.
Grete
G
Lottrup
Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark.
Kirstine
K
Belling
Department of Growth and Reproduction, Copenhagen University Hospital(Rigshospitalet), International Center for Research and Training in Endocrine Disruption of Male Reproduction & Child Health (EDMaRC), 9 Blegdamsvej, DK-2100 Copenhagen, Denmark.
Henrik
H
Leffers
Department of Growth and Reproduction, Copenhagen University Hospital(Rigshospitalet), International Center for Research and Training in Endocrine Disruption of Male Reproduction & Child Health (EDMaRC), 9 Blegdamsvej, DK-2100 Copenhagen, Denmark.
John E
JE
Nielsen
Department of Growth and Reproduction, Copenhagen University Hospital (Rigshospitalet), International Center for Research and Training in Endocrine Disruption of Male Reproduction & Child Health (EDMaRC), 9 Blegdamsvej, DK-2100 Copenhagen, Denmark.|Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark (DTU), DK-2800 Lyngby, Denmark.|DTU Multi-Assay Core (DMAC), Department of Biotechnology and Biomedicine, DTU Bioengineering, Technical University of Denmark, DK-2800 Lyngby, Denmark.
Marlene D
MD
Dalgaard
Department of Growth and Reproduction, Copenhagen University Hospital(Rigshospitalet), International Center for Research and Training in Endocrine Disruption of Male Reproduction & Child Health (EDMaRC), 9 Blegdamsvej, DK-2100 Copenhagen, Denmark.
Anders
A
Juul
Department of Growth and Reproduction, Copenhagen University Hospital(Rigshospitalet), International Center for Research and Training in Endocrine Disruption of Male Reproduction & Child Health (EDMaRC), 9 Blegdamsvej, DK-2100 Copenhagen, Denmark.
Niels E
NE
Skakkebæk
Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark.|Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark (DTU), DK-2800 Lyngby, Denmark.
Søren
S
Brunak
Department of Growth and Reproduction, Copenhagen University Hospital(Rigshospitalet), International Center for Research and Training in Endocrine Disruption of Male Reproduction & Child Health (EDMaRC), 9 Blegdamsvej, DK-2100 Copenhagen, Denmark.
Ewa
E
Rajpert-De Meyts
D000328:Adult; D055511:CCN Intercellular Signaling Proteins / Q000235:genetics / Q000378:metabolism; D016022:Case-Control Studies; D005333:Fetus; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D006911:Hydroxyprostaglandin Dehydrogenases / Q000235:genetics / Q000378:metabolism; D007985:Leydig Cells / Q000166:cytology / Q000378:metabolism*; D008297:Male; D009373:Neoplasms, Germ Cell and Embryonal / Q000235:genetics* / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D013091:Spermatogenesis / Q000235:genetics; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism; D013736:Testicular Neoplasms / Q000235:genetics* / Q000473:pathology; D059467:Transcriptome
D055511:CCN Intercellular Signaling Proteins; C413323:CCN5 protein, human; C406144:IGF2BP1 protein, human; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D006911:Hydroxyprostaglandin Dehydrogenases; D015238:Sulfotransferases; C028152:alcohol sulfotransferase
Leydig cell; Leydig cell hyperplasia; Leydig cell micronodules; foetal Leydig cell; gene expression profiling; human testis; testicular germ cell tumours; transcriptome