Six Decades of Research on Human Fetal Gonadal Steroids

Abstract

Human fetal gonads acquire endocrine steroidogenic capabilities early during their differentiation. Genetic studies show that this endocrine function plays a central role in the sexually dimorphic development of the external genitalia during fetal development. When this endocrine function is dysregulated, congenital malformations and pathologies are the result. In this review, we explain how the current knowledge of steroidogenesis in human fetal gonads has benefited from both the technological advances in steroid measurements and the assembly of detailed knowledge of steroidogenesis machinery and its expression in human fetal gonads. We summarise how the conversion of radiolabelled steroid precursors, antibody-based assays, mass spectrometry, ultrastructural studies, and the in situ labelling of proteins and mRNA have all provided complementary information. In this review, our discussion goes beyond the debate on recommendations concerning the best choice between the different available technologies, and their degrees of reproducibility and sensitivity. The available technologies and techniques can be used for different purposes and, as long as all quality controls are rigorously employed, the question is how to maximise the generation of robust, reproducible data on steroid hormones and their crucial roles in human fetal development and subsequent functions.

Keywords: androgens; detection; estrogens; fetal; human; ovary; quantification; steroidogenesis; testis.

Figure 1

Summary of the principal gonadal steroidogenic pathways. Steroid precursors and metabolites from the classic delta 4 (orange arrows) and delta 5 pathways (blue arrows) are shown together with the backdoor (alternative) pathway (green arrows).

Figure 2

The dynamic progression of steroidogenic cell populations in the human fetal testes and ovaries. Profiles of the numbers of steroidogenic testicular Leydig cells (blue line) and ovarian interstitial glandular cells (pink lines) are depicted in comparison with the differentiation of testicular and ovarian cords, respectively, and of testosterone circulating profiles (adapted from [6,7,8,9,10]). Post-conception week (PCW); Gestational week (GW); and days post conception (dpc).

Figure 3

A schematic explanation of the principal immunological assay types. All the immunological methods represented here rely on the use of a steroid-directed specific detection antibody fixed on a solid phase or on beads. The main difference lies in the detection technique. The steroid of interest-antibody complex will be detected even by a radioactive competitor (radioimmunoassay; RIA), or an enzymatic reaction (Enzyme Linked ImmunoSorbent Assay; ELISA), fluorescent dyes (Dissociation-enhanced lanthanide fluorescence immunoassay; DELFIA, Luminex technology), or chemiluminescent dyes (light-initiated chemiluminescent assays; LICA). The main components for each technology are described in the box.

Figure 4

Steroidomics workflow. Mass spectrometry analysis of steroids requires preliminary steps for the sample preparation. Steroids from human fetal gonads or culture medium are first extracted by liquid–liquid extraction (LLE) or solid-phase extraction (SPE). This step allows the concentration of the steroids and reduction of the biological matrix. The subsequent deconjugation and derivatization steps are necessary to, respectively, release the free steroid if needed and obtain chemical properties useful before the mass spectrometry analysis: good volatility, heat-resistance, and ionizability. Samples are then injected through the chromatographer (LC or GC) mounted upstream of the mass spectrometer to separate all steroids. The ionisation step (EI or ESI) transforms neutral steroids into mono or multicharged ions (z > 1), but only monocharged ions are analysed next. The sample preparation protocol and the ionisation method used depend on the chromatographic method. Then, ionised steroids are analysed according to their mass to charge ratio (m/z) by the tandem mass spectrometer analysers (MS2). Tandem mass spectrometers suitable for the study of steroids are Quadrupole-Time of Flight (Q-TOF), Q-Orbitrap, and Triple Quadrupole (QqQ). Due to their performances (high mass resolution, mass accuracy, and dynamic range) Q-TOF and Q-Orbitrap are mostly used in full scan mode and for non-targeted steroid profiling. In contrast, the QqQ instrument, a low-resolution mass analyser is used for targeted approaches. The final step of data processing depends on the tandem analysers used and the approach chosen (untargeted or targeted). Quantitative determination is absolute or relative, and steroids are identified following either database searches or comparison against high purity standards.